Non-Steroidal Anti-Inflammatory Drug Effects on Core Temperature

University of South Carolina Scholar Commons

Theses and Dissertations

6-30-2016 Non-Steroidal Anti-Inflammatory Drug Effects On Core Temperature, Hydration, Gastrointestinal Distress, And Performance In Exercising Humans Dawn Marie Emerson University of South Carolina

Follow this and additional works at: https://scholarcommons.sc.edu/etd Part of the Other Medicine and Health Sciences Commons

Recommended Citation Emerson, D. M.(2016). Non-Steroidal Anti-Inflammatory Drug Effects On Core Temperature, Hydration, Gastrointestinal Distress, And Performance In Exercising Humans. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/3402

This Open Access Dissertation is brought to you by Scholar Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. NON-STEROIDAL ANTI-INFLAMMATORY DRUG EFFECTS ON CORE TEMPERATURE, HYDRATION, GASTROINTESTINAL DISTRESS, AND PERFORMANCE IN EXERCISING HUMANS

Dawn Marie Emerson

Bachelor of Science The University of Alabama, 2006

Master of Science University of South Carolina, 2009

Submitted in Partial Fulfillment of the Requirements

For the Degree of Doctor of Philosophy in

Exercise Science

The Norman J. Arnold School of Public Health

University of South Carolina

2016

Accepted by:

J. Mark Davis, Major Professor

Toni M. Torres-McGehee, Committee Member

J. Larry Durstine, Committee Member

Stephen CL Chen, Committee Member

ii DEDICATION

To the Lord, Our God.

I would have nothing without Him. All joy, love, patience, and success in my life and surrounding this project is because of Him who blessed me. He is by my side in my weakness, turmoil, frustration, doubt, and anger. He does not give up, but challenges, encourages, and guides. I offer this and everything I do to Him.

Credo in unum Deum, Patrem omnipotentem, factorem caeli et terrae, visibilium omnium et invisibilium, Et in unum Dominum Iesum Christum, Filium Dei unigenitum, et ex Patre natum, ante omnia saecula, Deum de Deo, lumen de Lumine, Deum verum de Deo vero, genitum, non factum, consubstantialem Patri: per quem omnia facta sunt.

Qui propter nos homines et propter nostram salutem descendit de caelis. Et incarnatus est de Spiritu Sancto ex Maria Virgine, et homo factus est. Crucifixus etiam pro nobis sub Pontio Pilato; passus et sepultus est, et resurrexit tertia die, secundem Scripturas, et ascendit in caelum, sedet ad dexteram Patris. Et iterum venturus est cum gloria, iudicare vivos et mortuos, cuius regni non erit finis.

Et in Spiritum Sanctum, Dominum et vivificantem: qui ex Patre Filioque procedit.

Qui cum Patre et Filio simul adoratur et conglorificatur: qui locutus est per prophetas.

Et nuam, sanctam, catholicam et apostolicam Ecclesiam. Confiteor unum baptisma in remissionem peccatorum. Et exspecto resurrectionem mortuorum, et vitam venturi saeculi.

Amen.

iii ACKNOWLEDGEMENTS

Deepest appreciation to Dr. Mark Davis for mentoring me throughout my entire

time at USC. I would have never had this opportunity without his patience and willingness to educate. Thank you is hardly enough for Dr. Toni Torres-McGehee’s motivation, encouragement, mentorship, and friendship. Special thanks to Dr. Stephen Chen for his willingness to help in numerous capacities. Thank you to Dr. Larry Durstine for his time, expertise, humor, and easy going attitude. Immense gratitude to Dr. Brian Keisler for being

“on-call” and assisting through several challenges. Thank you to all the research assistants, especially Joe Bivona and Justin Stone for their commitment, brilliant minds, wit, and fortitude. Tremendous thanks to Craig Pfeifer; I could not have finished this without him.

Thank you participants. Thank you Riley Enos for spending hours pipetting and making concoctions. Much gratitude to Dr. Angela Murphy and Kei Lam at the School of

Medicine, Drs. William Cotham and Michael Walla and their team in the Department of

Biochemistry, and Jackson Murphy at Hawthorne Pharmacy. I thank the National Athletic

Trainers’ Association Foundation and the American College of Sports Medicine for funding portions of this study. I share my excitement with my parents, grandmothers, extended family, in-laws, and numerous friends (wish I could name you all). All of them had faith I could complete this endeavor, even if they did not understand what I was doing.

I cannot thank my husband, Charlie, enough. You have been my soundboard, helped in any way I asked, reassured me when I felt like giving up, celebrated all the high points, and

challenged me to be stronger. I could not have accomplished this without you!

iv ABSTRACT

Naproxen is a commonly used non-steroidal anti-inflammatory drug designed to relieve pain and inflammation. Due to potentially adverse effects on the gastrointestinal

(GI) tract, cardiovascular and immune system, and renal function, naproxen may negatively affect thermoregulation, fluid and electrolyte balance, and performance during exercise, particularly in the heat. Therefore, the purpose of this study was to determine the effects of naproxen on core temperature (Tc), inflammation, hydration, GI distress, and performance during cycling in a hot environment. We utilized a double-blind, randomized and counterbalanced, cross-over design to determine the effects of naproxen (dose = 3 220 mg naproxen sodium pills) or placebo (3 cellulose pills) on the dependent variables: Tc, interleukin-6 (IL-6), GI distress symptoms, fecal occult blood, plasma sodium and

potassium concentration, plasma osmolality, urine osmolality, urine specific gravity,

percent change in body mass, urine volume, fluid volume, heart rate, blood pressure, rate

of perceived exertion, and distance during a 10 min time trial. Participants (n = 11, age =

27.8 + 5.7 yrs, weight = 79.1 + 17.9 kg, and V̇ O2max = 41.4 + 5.7 mL/kg) completed 4

conditions: 1) placebo and ambient (Control); 2) naproxen and ambient (Npx); 3) placebo

and heat (Heat); and 4) naproxen and heat (NpxHeat) separated by a minimum of 7 days.

Dependent measures were taken pre-, during, post-, and 3 hrs post- a 90 min cycling protocol. We found no statistically significant differences between experimental conditions for any dependent variable. Exercise induced significant overall increases pre- to post- for

Tc, IL-6, plasma potassium, heart rate, blood pressure, perceived exertion, and GI distress.

v Compared to placebo, naproxen generally led to higher fluid intake and lower urine volume. Mean distance traveled was highest during Npx (3.3 + 0.8 miles) and lowest in

NpxHeat (2.8 + 0.8 miles). Participants experienced greater GI distress during Heat, especially day post-exercise. During exercise, GI symptom occurred in 64% of trials.

Interestingly, dizziness and headache were only reported during placebo conditions, with generally less systemic symptoms during exercise with naproxen use. In conclusion, our results do not support our hypotheses that naproxen would increase Tc, IL-6, and GI distress, alter fluid-electrolyte balance, and decrease performance compared to placebos.

vi TABLE OF CONTENTS

DEDICATION ...... iii

ACKNOWLEDGEMENTS ...... iv

ABSTRACT ...... v

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

LIST OF SYMBOLS ...... xii

LIST OF ABBREVIATIONS ...... xiii

CHAPTER 1 OVERALL INTRODUCTION ...... 1

CHAPTER 2 NAPROXEN EFFECTS ON CORE TEMPERATURE AND INFLAMMATION DURING CYCLING IN THE HEAT ...... 8

ABSTRACT ...... 9

INTRODUCTION ...... 10

METHODS ...... 11

RESULTS ...... 17

DISCUSSION ...... 19

REFERENCES ...... 25

CHAPTER 3 NAPROXEN EFFECTS ON HYDRATION AND ELECTROLYTE MEASURES DURING CYCLING IN THE HEAT ...... 36

vii ABSTRACT ...... 37

INTRODUCTION ...... 38

METHODS ...... 39

RESULTS ...... 45

DISCUSSION ...... 48

REFERENCES ...... 56

CHAPTER 4 NAPROXEN EFFECTS ON GASTROINTESTINAL DISTRESS AND PERFORMANCE DURING CYCLING IN THE HEAT ...... 62

ABSTRACT ...... 63

INTRODUCTION ...... 64

METHODS ...... 65

RESULTS ...... 71

DISCUSSION ...... 75

REFERENCES ...... 82

CHAPTER 5 PROPOSAL ...... 90

INTRODUCTION ...... 91

STATEMENT OF THE PROBLEM...... 92

SPECIFIC AIMS AND HYPOTHESES ...... 92

LITERATURE REVIEW ...... 93

METHODS ...... 153

REFERENCES ...... 164

BIBLIOGRAPHY ...... 188

APPENDIX A: INFORMED CONSENT FORM ...... 212

APPENDIX B: HEALTH AND INJURY HISTORY QUESTIONNAIRE ...... 219

viii APPENDIX C: TAKE HOME INSTRUCTIONS ...... 223

C.1. FIRST TRIAL E-MAILS 4 DAYS PRE-DATA COLLECTION ...... 223

C.2. REMINDER EMAIL DIET AND ACTIVITY LOG ...... 224

C.3. SUBSEQUENT TRIALS E-MAILS 4 DAYS PRE-DATA COLLECTION ...... 224

C.4. PRE-DATA COLLECTION TAKE HOME INSTRUCTIONS ...... 225

C.5. POST-DATA COLLECTION TAKE HOME INSTRUCTIONS ...... 226

APPENDIX D: GASTROINTESTINAL SYMPTOMS INDEXES ...... 228

D.1. GI SYMPTOM INDEX DURING EXERCISE ...... 228

D.2. GI SYMPTOM INDEX PRE- AND POST- EXERCISE ...... 229

APPENDIX E: DATA COLLECTION PROCEDURE CHECKLISTS ...... 230

E.1. INFORMATION SESSION ...... 230

E.2. V̇ O2MAX TESTING ...... 230

E.3. PRE-TRIAL APPOINTMENT...... 232

E.4. POST-TRIAL APPOINTMENT ...... 233

E.5. DATA COLLECTION SESSION ...... 233

APPENDIX F: ADDITIONAL TABLES AND FIGURES ...... 240

ix LIST OF TABLES

Table 2.1 Participant Demographics ...... 29

Table 3.1 Hydration Measures, Fluid Volume, and Urine Volume for Experimental Conditions ...... 59

Table 3.2 Plasma Electrolytes for Experimental Conditions ...... 60

Table 4.1 Participant Demographics ...... 86

Table 4.2 Performance Measures during Cycling for Experimental Conditions ...... 87

Table 4.3 Mean and Maximum Gastrointestinal Symptom Scores for Experimental Conditions ...... 88

Table 4.4 Mean and Maximum Gastrointestinal Symptom Scores for Experimental Conditions during Exercise ...... 89

Table F.1. Fluid Volume and Urine Volume for Gender by Experimental Conditions ...... 245

Table F.2. Diet and Exercise Overall and for Gender...... 247

Table F.3. Rate of Perceived Exertion during Cycling for Gender by Experimental Conditions ...... 248

Table F.4. Distance and Max Heart Rate during Cycling for Gender by Experimental Conditions ...... 249

Table F.5. Mean, Maximum, and Serious (> 4) Gastrointestinal Symptom Scores for Experimental Conditions ...... 250

Table F.6. Mean and Serious (> 4) Gastrointestinal Symptom Scores for Experimental Conditions during Exercise ...... 251

x LIST OF FIGURES

Figure 2.1 Mean Core Temperature during Cycling and Rest for Experimental Conditions ...... 30

Figure 2.2a-d Regression and Participant Core Temperature during Cycling ...... 31

Figure 2.3 Mean IL-6 Pre-, Post-, and 3 Hrs Post-Cycling for Experimental Conditions ...... 35

Figure 3.1 Experimental Procedures Schematic ...... 61

Figure 5.1 Working Model for Overall Study ...... 186

Figure 5.2 Experimental Procedures Schematic ...... 187

Figure F.1 Male Mean Core Temperature during Cycling and Rest for Experimental Conditions ...... 240

Figure F.2 Female Mean Core Temperature during Cycling and Rest for Experimental Conditions ...... 241

Figure F.3 Mean Core Temperature during Cycling and Rest for Gender ...... 242

Figure F.4 Mean IL-6 Pre-, Post-, and 3 Hrs Post-Cycling for Males ...... 243

Figure F.5 Mean IL-6 Pre-, Post-, and 3 Hrs Post-Cycling for Females ...... 244

Figure F.6 Mean Systolic BP Pre-, Post-, and 3 Hrs Post-Exercise for Experimental Conditions by Gender ...... 246

xi LIST OF SYMBOLS

α alpha

β beta

χ2 chi-squared

°C degree Celsius

= equal

λ gamma

< less than

% percent

+ plus and minus

xii LIST OF ABBREVIATIONS

APC ...... antigen presenting cell

ADH ...... anti-diuretic hormone

ATP ...... adenosine triphosphate

BP ...... blood pressure

BM ...... body mass

bpm ...... beats per minute

cAMP ...... cyclic adenosine monophosphate

CI...... confidence interval

CNS ...... central nervous system

Control ...... placebo and ambient environment

COX ...... cyclooxygenase

Cr-EDTA...... chromium-labelled ethylenediaminetetra-acetate

Da ...... Dalton

DNA ...... deoxyribonucleic acid eg ...... for example

EHI ...... exertional heat illness

EHS ...... exertional heat stroke

EP ...... E prostanoid receptor

FIT...... fecal immunological test

xiii Fvol ...... fluid volume g...... gram g-FOBT ...... guaiac-based fecal occult blood test

GI ...... gastrointestinal

GSH...... glutathione

Heat ...... placebo and hot environment hr ...... hour

HR ...... heart rate

HSP ...... heat shock protein ie ...... in other words

IL ...... interleukin kg...... kilogram

L ...... liter

L/M ...... lactulose mannitol ratio

LPS ...... lipopolysaccharide min ...... minute mg ...... milligram ml ...... milliliter mmol ...... millimol mOsm ...... milliosmoles mph ...... miles per hour mSv ...... millisievert n...... sample size

xiv ng...... nanogram

NKC ...... natural killer cell

Npx ...... naproxen and ambient environment

NpxHeat ...... naproxen and hot environment

NSAID ...... non-steroidal anti-inflammatory drug

PEG ...... polyethylene glycol

PG ...... prostaglandin pH ...... acid base balance

PK ...... plasma potassium concentration

PNa ...... plasma sodium concentration

PK ...... prostaglandin

Posm ...... plasma osmolality pg...... picogram

RH ...... relative humidity

RNA ...... ribonucleic acid

ROS ...... reactive oxygen species

RPE ...... rate of perceived exertion

RR ...... relative risk

SIR ...... systemic inflammatory response

T4 ...... helper T lymphocyte

T8 ...... cytotoxic T lymphocyte

Tc ...... core temperature

TNF ...... tumor necrosis factor

xv Treg ...... regulatory T cells

TX ...... thromboxane

Uosm ...... urine osmolality

Usg ...... urine specific gravity

Uvol...... urine volume

V̇ O2max ...... maximum oxygen consumption

V̇ O2peak ...... peak oxygen consumption yr ...... year

xvi CHAPTER 1

OVERALL INTRODUCTION

Intense exercise places strain on multiple physiological systems that work to maintain core temperature (Tc), cardiovascular function, and fluid and electrolyte balance.

When these systems become less efficient, individuals present with signs and symptoms associated with health and performance decrements. For example, when the body fails to thermoregulate, Tc increases. If this increase occurs beyond a certain point, typically above

40°C, cells begin to die and a systemic inflammatory response (SIR) can ensue, leading to organ failure and death.1,2 Therefore, when thermal stress is added to intense exercise the demand on these systems increases.

Importantly, the thermoregulatory, cardiovascular, and fluid-electrolyte systems work concurrently to maintain homeostasis. Changes in these three systems often depend on exercise intensity. In general, the cardiovascular system responds by shunting blood, approximately 80%, away from the gastrointestinal (GI) organs to working muscles.3 As exercise continues, the cardiovascular system will maintain blood pressure (BP) and cardiac output by increasing heart rate (HR). At the same time, working muscles are producing a large amount of metabolic heat, which increases Tc. To mitigate this, blood is shunted to peripheral vessels to dissipate heat through the skin.3 If an individual maintains hydration the cardiovascular system is more capable of keeping up with the exercise and/or thermal demands. However, when dehydration occurs plasma volume decreases, which

stimulates the renin-angiotensin-aldosterone system to release hormones (ie, aldosterone

and vasopressin) that act on the kidneys and blood vessels. In the kidneys, aldosterone

causes sodium reabsorption. As a powerful cation, sodium causes water to be reabsorbed,

subsequently increasing plasma volume and BP.4 In a similar way, vasopressin, also known

as anti-diuretic hormone, causes kidneys to reabsorb water.3 Finally, blood continues to be

shunted from non-working muscles and the GI through vasoconstriction, which assists in increasing BP.1 As long as the body’s systems can maintain function, an individual can

continue exercise until he/she voluntarily stops. When one or more of these systems begin

to fail the person either continues exercise while experiencing signs and symptoms (eg,

headache, tachycardia, fatigue, abdominal cramping) or the person involuntarily stops

exercise (eg, loss of consciousness, cardiac failure).

Indications for thermoregulatory failure may present as an exertional heat illness

(EHI), such as heat syncope, heat exhaustion, or, in extreme cases, heat stroke (EHS).

Milder symptoms, typically seen with syncope or exhaustion, include lightheadedness,

dizziness, fatigue, nausea, and headache.5 Cardiovascular failure often accompanies EHS.

Individuals present with more serious symptoms, such as central nervous system

dysfunction (ie, loss of consciousness, irritability, or irrational behavior), multi-organ

failure, or death.5

Fluid-electrolyte imbalances during intense exercise and thermal strain include

hypohydration, hyperkalemia (plasma potassium > 5 mmol/L),6 and hyponatremia (plasma

sodium concentration < 135 mmol/L).7 One big concern for hypohydration is how it affects

the other systems. Symptoms for hypohydration vary on severity (mild to extreme

hypohydration), and are similar to those of EHIs (ie, dizziness, headache, fatigue).

Hypohydration, as little as 2% body mass loss, can perpetuate Tc increase because of

increased cardiovascular strain.8,9 Elevated plasma potassium during exercise primarily comes from working skeletal muscle releasing potassium.6 When systems are functioning

appropriately, particularly the kidneys, potassium can be cleared from the blood before

detrimental effects occur. This additionally supports the need to maintain hydration in order

to maintain renal function, since hyperkalemia can lead to extreme cardiac failure (ie,

stroke and heart attack).6 Sodium is essential for many physiological functions. When

plasma sodium levels are depleted an individual may experience many of the same

symptoms of hypohydration and EHIs (ie, headache, dizziness, weakness, and nausea).

During severe hyponatremia, the excessive water intake and dramatic drop in sodium can

lead to cerebral and pulmonary edema, central nervous system dysfunction, seizure, and

death.7 The primary etiology for hyponatremia is consuming hypotonic fluids in excess of

fluid lost through sweat and respiration. Hyponatremia can also be induced from

inappropriate release of vasopressin, causing water retention and diluting sodium, or

excessive sodium loss through sweat.7

Together, the cardiovascular, thermoregulatory, and fluid-electrolyte systems are critical in maintaining health and performance during activity and can be influenced by two other important systems – the GI and immune. Major effects on the GI during intense exercise include hypoxia from shunted blood, disruption in epithelial barrier integrity,10

and direct damage from mechanical vibrations and increased abdominal pressure.11 These

can upregulate immune responses and lead to SIR. As previously mentioned, SIR is seen

during EHS. The similarities between the two has led to a closer examination of EHS

etiology, going beyond the early theory of cells simply becoming “too hot”, to what now

3 some consider a dual model for developing EHS.1 This model is highly based on influences from both the immune system and GI tract. The first model suggests hyperthermia is caused by exercise in the heat and exacerbated by GI ischemia, inflammatory cytokine release, and endotoxin release. Exercise continues until the body is overloaded by endotoxin and pyrogenic cytokines, which cause cell necrosis, decreased plasma volume, fever, and eventually SIR and EHS.1 The second model suggests that regardless of environment, prolong intense exercise suppresses the immune system. The individual, exercising with a blunted immune response and/or subclinical infection, exercises but can no longer handle the demands to maintain cardiac function and Tc. Inflammatory responses drive endotoxin release from the GI and, like the first model, SIR and EHS can occur.1 This latter model helps explain why some individuals can experience EHS in cooler environmental temperatures12 or with Tc below 40.5°C.

Presumably, any factor that can perpetuate cardiovascular failure, thermoregulatory failure, fluid-electrolyte imbalance, or increase immune and GI responses can predispose an individual to developing EHI. There are a number of known intrinsic and extrinsic risk factors for EHS. Extrinsic variables (ie, equipment, clothing, rest breaks, nutrition, water access)5 are more easily controlled. On the other hand, intrinsic variables are more difficult to understand and may be unknown to the individual or healthcare professionals working with physically active individuals. For instance, previous or current illness can elicit an immune response causing subclinical fever and inflammation, which would decrease thermal tolerance.

Some medications stimulate metabolic heat production or alter cardiovascular function and subsequently fluid-electrolyte balance.5 Non-steroidal anti-inflammatory

drugs (NSAIDs) are not currently considered a risk factor for EHS. This is primarily

attributed to their association with being anti-pyretic and anti-inflammatory. Another reason is that existing literature results are conflicting. It is difficult to make conclusions because of the wide variety of NSAIDs available and the doses used. In theory and despite their anti-pyretic and anti-inflammatory affects, NSAIDs may be a risk factor due to the adverse effects they have on the GI epithelial lining, cardiovascular system, and renal function. Regardless of the type of NSAID, research has established they directly damage the GI, causing ulcers, hemorrhaging, and increased barrier permeability.13,14

Gastrointestinal damage causes an inflammatory response, upregulating cytokine release.15

NSAIDs promote vasoconstriction in both the kidneys and peripheral blood vessels. Renal

vasoconstriction decreases urine volume by retaining fluid, which dilutes plasma sodium.

Peripheral vasoconstriction decreases the effectiveness to eliminate metabolic heat production, increasing Tc. Finally, both vasoconstriction and fluid retention increase

BP.7,16 It can be presumed, through these adverse effects, NSAIDs have several pathways

to promote either model of EHS.

The little research available and conflicting results makes it difficult to determine

whether NSAIDs are risk factors for EHS. Some studies have found NSAIDs have no effect

on Tc during exercise,17,18 while others have found they increase Tc.19 No research has examined naproxen’s effects on Tc. Presently very little research on NSAIDs using both males and females and comparing ambient and hot conditions exists. The purpose of this

study was to contribute to the body of literature in this area by examining naproxen on thermoregulation, cardiovascular function, and fluid-electrolyte balance during exercise.

Identifying and understanding potential risk factors for EHS may increase awareness and

5 improve prevention techniques for physically active persons and those working with these individuals

REFERENCES

1. Lim CL, Mackinnon LT. The roles of exercise-induced immune system disturbances in the pathology of heat stroke : the dual pathway model of heat stroke. Sports Med. 2006;36(1):39-64.

2. Berg A, Muller H-M, Rathmann S, Deibert P. The gastrointestinal system - an essential target organ of the athlete's health and physical performance. Exerc Immunol Rev. 1999;5:78-95.

3. McArdle WD, Katch FI, Katch VL. Exercise Physiology: Energy, Nutrition, and Human Performance. Fifth ed. Baltimore, MD: Lippincott Williams & Wilkins; 2001.

4. Johnson AL, Criddle LM. Pass the salt: indications for and implications of using hypertonic saline. Crit Care Nur. 2004;24(5):36-48.

5. Casa DJ, DeMartini JK, Bergeron MF, et al. National Athletic Trainers' Association position statement: exertional heat illnesses. J Athl Train. 2015;50(9):986-1000.

6. Clausen T. Hormonal and pharmacological modification of plasma potassium homeostasis. Fundam Clin Pharmacol. 2010;24(5):595-605.

7. Rosner MH, Kirven J. Exercise-associated hyponatremia. Clin J Am Soc Nephrol. 2007;2(1):151-161.

8. Maxwell NS, Gardner F, Nimmo MA. Intermittent running: muscle metabolism in the heat and effect of hypohydration. Med Sci Sport Exerc. 1999;31(5):675-683.

9. Casa DJ, Stearns RL, Lopez RM, et al. Influence of hydration on physiological function and performance during trail running in the heat. J Athl Train. 2010;45(2):147-156.

10. Carden DL, Granger DN. Pathophysiology of ischemia-reperfusion injury. J Pathol. 2000;190:255-266.

11. de Oliveira EP, Burini RC. The impact of physical exercise on the gastrointestinal tract. Curr Opin Clin Nutr Metab Care. 2009;12(5):533-538.

12. Roberts WO. Exertional heat stroke during a cool weather marathon: a case study. Med Sci Sports Exerc. 2006;38(7):1197-1203.

13. Gibson GR, Whitacre EB, Ricotti CA. Colitis induced by nonsteroidal anti- inflammatory drugs. Arch Intern Med. 1992;152:625-632.

14. Lambert GP, Schmidt A, Schwarzkopf K, Lanspa S. Effect of aspirin dose on gastrointestinal permeability. Int J Sports Med. 2012;33(6):421-425.

15. Appleyard CB, McCafferty D-M, Tigley AW, Swain MG, Wallace JL. Tumor necrosis factor mediation of NSAID-induced gastric damage: role of leukocyte adherence. Am J Physiol Gastrointest Liver Physiol. 1996;270:G42-G48.

16. Garella S, Matarese RA. Renal effects of prostaglandins and clinical adverse effects of nonsteroidal anti-inflammatory agents. Medicine. 1984;63(3):165-181.

17. Jacobson ED, Bass DE. Effects of sodium salicylate on physiological responses to work in heat. J Appl Phys. 1964;19(1):33-36.

18. Downey JA, Darling RC. Effect of salicylates on elevation of body temperature during exercise. J Appl Phys. 1962;17(2):323-325.

19. Bruning RS, Dahmus JD, Kenney WL, Alexander LM. Aspirin and clopidogrel alter core temperature and skin blood flow during heat stress. Med Sci Sports Exerc. 2013;45(4):674-682.

CHAPTER 2

NAPROXEN EFFECTS ON CORE TEMPERATURE AND INFLAMMATION DURING EXERCISE IN

THE HEAT1

1Dawn M. Emerson, J. Mark Davis, Toni M. Torres-McGehee, Stephen CL Chen, J. Larry Durstine, Justin V. Stone, Joseph D. Bivona, Charles C. Emerson, Craig E. Pfeifer. To be submitted to Med Sci Sports Exerc.

ABSTRACT

Purpose: Existing literature regarding non-steroidal anti-inflammatory drugs (NSAIDs) on core temperature (Tc) during exercise is limited and conflicting. Our aim was to determine the effects of naproxen, a common NSAID, on Tc and interleukin-6 (IL-6) in hydrated, exercising humans. Methods: We utilized a double-blind, randomized and counterbalanced, cross-over design to determine effects of a 24 hr naproxen dose (220 mg naproxen/dose) and placebo (0 mg naproxen/dose) on Tc and IL-6 during cycling in a hot or ambient environment. Participants (n = 11; 6 male, 5 female) completed 4 conditions:

1) placebo and ambient (Control); 2) placebo and heat (Heat); 3) naproxen and ambient

(Npx); and 4) naproxen and heat (NpxHeat). Participants cycled for 90 min then rested 3 hrs in an ambient environment. Throughout exercise, participants drank water (3.5 ml/kg) every 15 min to maintain euhydration. Dependent measures were taken pre-, during, post- and 3 hrs post-cycling. Results: Overall Tc significantly increased pre- to post-cycling

(37.1 + 0.4°C to 38.2 + 0.3°C, P < 0.001) and decreased during rest (P < 0.001), but neither

rate of change or maximum Tc were significantly different between conditions. IL-6

increased pre- to post-exercise (0.54 + 0.06 pg/ml to 2.46 + 0.28 pg/ml, P < 0.001), and

remained significantly higher than pre- at 3 hrs post- (1.17 + 0.14 pg/ml, P = 0.001). No

significant IL-6 differences were found between conditions. Conclusion: A 24 hr dose of

over the counter strength naproxen did not significantly affect Tc or IL-6 among hydrated

males and females cycling in hot and ambient conditions. Key Words: cytokine, exertional

heat illness, NSAIDs, thermoregulation

INTRODUCTION

Intense physical activity and/or thermal stress is known to induce gastrointestinal

(GI) damage and a number of inflammatory responses.(8) Damage to the GI epithelial barrier lining increases GI permeability and causes inflammatory cytokine release.(6,8)

Maintaining GI barrier integrity mitigates potentially toxic products, such as bacterial lipopolysaccharide (ie, endotoxin), from moving from the gut into blood circulation. When integrity is compromised these products can enter the blood and cause severe conditions, such as endotoxemia and systemic inflammatory response (SIR).(8)

Symptoms associated with SIR are similar to those seen with exertional heat illness

(EHI), particularly exertional heat stroke (EHS). For example, both EHS and SIR can present with vomiting, dehydration, cell necrosis, cardiovascular compromise, and multi- organ failure.(7,8) Additionally, both conditions can be driven by GI distress or compromised immune function. An individual exercising with an underlying inflammatory response is at greater risk for EHI due to thermal intolerance.(8)

Exertional heat stroke is identified by two key characteristics: 1) a Tc > 40.5°C and

2) altered central nervous system function.(3) It is important to note that 40.5°C is not a

“golden number”. It remains elusive why some individuals can participate in intense activity with Tc above 40.5°C without developing EHS, and other individuals experience

EHS below this temperature. Extensive scientific literature has examined risk factors,(1,5) ways to prevent,(2,3) and ways to treat EHS.(4,9) However, preventable EHS deaths still occur. Individual variability among EHS cases suggests there are a number of less

understood intrinsic factors. Additional extrinsic variables, such as non-steroidal anti- inflammatory drugs (NSAIDs), may also play a role.

NSAIDs reduce fever, inflammation, and pain by inhibiting cyclooxygenase (COX) derived prostaglandins (PGs) that induce these responses. However, NSAIDs are also associated with a number of adverse effects on the GI and immune systems. These adverse events are closely associated with developing SIR and EHIs. Like intense exercise and thermal stress, NSAIDs increase GI permeability,(12,13,25) cause GI distress(6,14) and induce inflammatory cytokines.(17,28,32) Despite these similarities, limited research has

evaluated NSAID effects on Tc during exercise, particularly during thermal stress. Older

studies found sodium salicylate(11) and aspirin(7) had no effect on Tc compared to

placebos. However, a more recent investigation using aspirin showed significant Tc

increases due to blunted skin blood flow.(4) Our study sought to determine acute dose

effects of a commonly used over the counter NSAID, naproxen, on Tc and the

inflammatory cytokine interleukin-6 (IL-6). We hypothesized naproxen would increase Tc

and plasma IL-6 levels significantly during exercise in the heat compared to placebo

controls.

METHODS

Participants

Eleven moderately trained, healthy, non-heat acclimatized participants (6 male, 5

female) were recruited from the university and surrounding community to complete this

study. Participant demographical information is presented in Table 2.1. Prior to

participation, participants read and signed an Institutional Review Board approved

informed consent form (Appendix A). Inclusion criteria was determined by a health and

injury history questionnaire (Appendix B) and V̇ O2max test (Appendix E.2). Participants

were free from cardiovascular, respiratory, and metabolic disorders; musculoskeletal

disorders preventing cycling exercise; fluid and electrolyte balance disorders; and GI and

swallowing disorders. Participants were also asked about current over the counter and

prescription medication use to ensure no potentially harmful interactions. Any participant

currently taking analgesic or anti-inflammatory medication was asked to cease the

medication for the study duration. Participants were asked about previous EHI history, but

this was not an exclusion criteria. Lastly, females completed questions regarding menstrual

cycle. Participants completed a graded cycling V̇ O2max test to ensure they were moderately trained (male V̇ O2max between 35-40 mL/kg and female V̇ O2max between 32-40

mL/kg).(21)

Experimental Conditions

Four experimental conditions were completed in a randomized, counter-balanced

order. A minimum of 7 days separated each trial. Prior to beginning exercise, participants

took a 24 hr dose (3 capsules) of placebo or naproxen. Participants consumed 1 capsule at

16 hrs, 8 hrs, and 0 hrs before data collection. All participants were given specific take

home instructions to follow 24 hrs prior to data collection, including directions for taking

each capsule with 1 8oz glass of water and not with food. All trials took place in an

environmental chamber (hot = 35.7 + 1.3°C, 53.2 + 3.2% relative humidity [RH]) or

laboratory (ambient = 22.7 + 1.8°C, 52.4 + 5.5% RH).

Control. Participants in the Control condition were given 3 placebo capsules

(cellulose) and exercised in an ambient environment.

Naproxen (Npx). Participants in the Npx condition were given 3 naproxen capsules

(220 mg naproxen sodium/dose) and exercised in a hot environment.

Placebo and Heat (Heat). Participants in the Heat condition were given 3 placebo

capsules (cellulose) and exercised in a hot environment.

Naproxen and Heat (NpxHeat). Participants in the NpxHeat condition were given

3 naproxen capsules (220 mg naproxen sodium/dose) and exercised in a hot environment.

Instruments and Protocols

Anthropometric Measures. Weight and body fat percentage was measured using a bioelectrical impedance analysis scale (Tanita SC-331S Body Composition Monitor,

Tanita Co., Tokyo, Japan). Height was self-reported.

Health and Injury History Questionnaire (Appendix B). Details in Chapter 3.

Cycle Exercise Protocol. Participants completed a 90 min cycle protocol on a stationary bike (Monark Ergomedic 828E, Monark Exercise AB, Vansbro, Sweden).

Before starting a 3 min warm-up, participants were provided their target heart rate (HR) corresponding to 70% V̇ O2max to maintain during 80 min of cycling.(29) Heart rate was

monitored using Polar HR monitors (Polar Electro Inc., Lake Success, NY). Monitors were

within the participant’s visual and researchers provided verbal cues to assist participants in

maintaining HR throughout. Following 80 min, participants cycled 10 min at maximum

effort. Research assistants gave each participant verbal encouragement. The exercise

protocol ended with a 5 min cool down.

Inflammatory Cytokines. We collected blood from a cubital vein pre-, post-, and

3 hrs post-exercise. Two 6 ml vials were collected into a vacutainer tube and inverted

several times to mix. We assessed IL-6 using enzyme linked immunosorbent assay high

13 sensitivity kits (R&D Systems human IL-6 Quantikine ELISA kit, R&D Systems, Inc.,

Minneapolis, MN).

Core Temperature. Core temperature was measured using rectal thermometry

(Doric 450 Series digital thermometer, VAS Engineering, Inc., San Diego, CA).

Participants were instructed to insert the probe 10 cm past the anal sphincter. During exercise, Tc was continuously monitored and recorded every 5 min. Participants were not allowed to exceed a Tc > 40°C. During the 3 hr rest period, Tc was recorded at 5 and 10 min post-exercise, then every 15 min until the participant reached baseline (pre-exercise

Tc) or the rest period concluded.

Hydration. Participants were required to be euhydrated prior to beginning exercise.

To maintain hydration during exercise, participants were provided 3.5 ml/kg of water to consume every 15 minutes. Hydration status was measured using urine specific gravity

(Usg) by a handheld clinical refractometer (model REF 312, Atago Company Ltd., Tokyo,

Japan). Euhydration was defined as Usg < 1.020.(26) A clean catch method was used in which the participant was instructed to urinate a small amount before placing the specimen cup in midstream to collect a minimum of 1 ounce of urine. Hydration measures were taken pre-, post-, and 3 hrs post-exercise.

Diet and Activity Logs. To control for pre-data collection dietary and physical activity influences, participants were asked to track diet and physical activity for 3 days prior and 1 day after data collection using an online nutrition software FoodProdigyTM

(ESHA Research, Salem, OR). Participants were encouraged to maintain similar diet and physical activity habits during the 24 hrs prior to each data collection session.

Experimental Procedures

Information Session. After all inclusion criteria were met, participants were

randomly assigned to experimental conditions by a research assistant. Participants, primary

investigators, and primary research assistants were blinded to which condition (naproxen

or placebo) participants were completing. Participants were familiarized with the online

diet and activity log. The V̇ O2max test was used to familiarize participants with the

stationary cycle. Seat and handle bar position were noted to maintain consistency

throughout trials.

Pre-Data Collection. Seventy-two hrs prior to data collection, participants were

sent an email with instructions for completing the diet and physical activity log. Logs

included the 72 hrs before and 24 hrs after data collection. Participants were also instructed

to refrain from intense, vigorous exercise for 48 hrs pre-trial.(22) Twenty-four hrs prior, participants received written take-home instructions and information was reviewed verbally by a research assistant. Instructions included when to take 2 capsules (placebo or naproxen), refrain from consuming alcohol and exercising, and to maintain normal sleep behaviors. Participants were also instructed to consume fluids, such as water, to ensure euhydration upon arriving for data collection. Lastly, participants were instructed to consume a small meal at least 2 hrs before arriving to ensure no food was in the stomach.

Data Collection Session. Once at the laboratory, participants were verbally asked if they took the 2 pills before taking the 3rd pill. Baseline HR, Tc, and Usg were measured

prior to blood collection. During the 90 min cycling, participants consumed water every 15

min, and researchers continuously monitored Tc and HR. At the conclusion of the 10 min

maximum effort, participants cooled down on the bike for 5 min. Participants then rested

for 5 min where post-blood was collected. Participants provided a urine sample for

hydration assessment. During the 3 hr rest period, participants sat in a semi-reclined/seated

position in an ambient environment (23°C and 56% RH). Each participant received a

standardized non-sucrose snack based on individual weight and were allowed to consume

water ad libitum. Throughout the rest, Tc was recorded every 15 min. Blood and urine

measures were collected at the rest conclusion. Before leaving, take home instructions were

reviewed with the participant regarding the 1 day post diet and activity log.

Statistical Analysis

IBM SPSS Statistics (version XXII; IBM Corporation, Armonk, NY) was used for

all analyses. Descriptive statistics (mean and standard deviations) for all dependent

variables were calculated. A one-way ANOVA assessed differences in demographics between conditions and gender (eg, age, height, weight, body fat %). Changes in Tc were

assessed using regression models and two repeated measures ANOVAs: 1) 4 (condition) x

20 (cycling) and 2) 4 (condition) x 9 (rest). A 4 (condition) x 3 (time) repeated measures

ANOVA was used to determine differences in pre-, post-, and 3 hr post-cycling IL-6 and

Usg. The same repeated measures ANOVAs were conducted to determine differences in

Tc and IL-6 between conditions within genders. Differences for overall Tc between genders was assessed using 2 (gender) x 20 (cycling) and 9 (rest) repeated measures

ANOVAs. A 2 (gender) x 3 (time) repeated measures ANOVA determined differences in

IL-6. Assumptions of sphericity were verified to determine whether variance in the differences within experimental conditions were equal or significantly different.

Greenhouse-Geisser corrections were used for Tc and IL-6 when sphericity was violated.

Post hoc analysis was conducted on significant main effects with Bonferroni corrections.

Power calculations were conducted a priori using standard deviations for Tc and IL-6. A

sample size of 8 was necessary to achieve a statistical power of 0.8. Our participant number

(n =11) is consistent with previously published research.(9,11,12,25,29) Significance level

was set at P < 0.05 for all analyses.

RESULTS

Sixteen participants began the study. Two participants dropped due to the intensity

of the study and 3 dropped due to the time commitment, yielding 11 participants. We

identified significant gender differences for height and body fat % (Table 2.1), but no other

demographical measures were significantly different. Five of 6 female participants used

oral contraceptives. Only one participant reported a previous history of EHI. All

participants began experimental trials euhydrated (Usg = 1.012 + 0.005) and remained

euhydrated to post- (Usg = 1.011 + 0.008) and 3 hr post-exercise (Usg = 1.007 + 0.006).

No significant Usg differences existed between experimental conditions at any time point.

Dietary analysis revealed no significant differences in calorie, carbohydrate, protein, sodium, or fat intake between conditions. Physical activity level 24 hrs before remained low to sedentary and was not significantly different between conditions.

Core Temperature

Figure 2.1 shows Tc changes throughout exercise and rest for each experimental

condition. Overall, Tc significantly increased from pre- (37.1 + 0.4°C) to post-exercise

(38.2 + 0.3°C, P < 0.001). During rest, Tc significantly decreased over time (P < 0.001)

and reached pre-exercise by 75 min post-exercise. Starting at 60 min cycling, mean Tc was higher in heat trials compared to ambient (Figure 2.1) and remained higher throughout rest.

However, there were no statistically significant Tc differences between experimental

conditions at any time point.

Figures 2.2.a-d illustrate individual participants’ Tc, overall mean, and regression

statistics for each condition during exercise. During Control and Npx, Tc increased

curvilinear during cycling, with a cubic regression model providing the best fit. Both Heat

and NpxHeat followed a linear model with no difference in slope between conditions.

Similar results were found for Tc during rest, with a curvilinear, cubic model providing the

best fit for all conditions and no difference in slope between conditions.

Regarding gender, a significant main effect was found for Tc change over time within males (P < 0.003) and females (P < 0.001). For males we found no significant Tc differences between conditions (Appendix F, Figure F.1); however, the main effect for Tc and condition during exercise approached significance (P = 0.075). Within females there was no significant differences between conditions at any time point (Appendix F, Figure

F.2). When combined (Appendix F, Figure F.3.), pre-exercise mean female Tc was significantly higher (37.3 + 0.3°C) than males (37.0 + 0.4°C, P = 0.02). During cycling,

females stayed significantly higher until 15 min. During rest, male Tc was significantly

higher at 10 min post-cycling (38.3 + 0.5°C versus 38.0 + 0.4°C, P < 0.02). Females were

significantly higher again at 75 post-cycling (37.3 + 0.3°C versus 37.1 + 0.4°C, P = 0.006)

and remained higher until the end of rest (37.3 + 0.2°C versus 36.8 + 0.3°C, P < 0.001).

Cytokines

Differences in IL-6 between pre-, post-, and 3 hrs post-exercise are presented in

Figure 2.3. No significant differences were found between conditions at any time point. A

significant change in mean IL-6 occurred over time (P < 0.001), with post-exercise IL-6

significantly higher than pre- and 3hrs post- for all conditions. At 3 hrs post-exercise, both

Npx (1.06 + 0.73 pg/ml) and Heat (0.97 + 0.49 pg/ml) were significantly higher compared

to pre- (Npx and Heat mean = 0.44 + 0.29 pg/ml, P < 0.04).

A significant main effect for IL-6 was found across time for gender (P = 0.001).

Additional analysis found no significant difference between genders at any time point between conditions. Within males (Appendix F, Figure F.4) there was a significant main effect over time within each condition (P < 0.04). Post hoc analysis showed only Heat and

NpxHeat were significantly greater post-exercise compared to pre-. No other significant differences were found for males. Significant main effects were found within Control and

Npx for females (Appendix F, Figure F.5). Post-exercise IL-6 was significantly greater than pre- in the Npx condition. Though not statistically significant, mean post-exercise IL-

6 during NpxHeat was higher than all other conditions and remained elevated 3 hrs post- exercise.

DISCUSSION

Due to anti-pyretic effects, NSAIDs have been theorized to decrease or blunt Tc

rise during exercise. However, existing literature has been unable to find NSAIDs elicit

this effect, instead finding they cause no change(7,11) or may increase Tc.(4) Our study

sought to determine whether a 24 hr over the counter dose of naproxen would negatively

affect thermoregulation and inflammation during moderate-intense cycling. Compared to

a placebo, our results indicated naproxen did not significantly affect Tc or IL-6 in either an

ambient or hot environment. These results are consistent with males walking in a hot

environment using 7.8 g sodium salicylate(11) and in ambient conditions using aspirin

acutely (1½ hrs pre-exercise) or chronically (daily doses for 2 and 7 days).(7) Sodium

salicylate is generally considered less effective than aspirin at inhibiting PGs and is not

known for its antipyretic effects.(2) Therefore, it is not surprising taking sodium salicylate

would not elicit significant Tc changes.

Lack of significance in max Tc and rate of Tc increase between conditions may be

attributed to our dosage. Using low dose aspirin (81 mg daily for 7 days) in older males

and females who rested and then cycled 2 hrs at 60% V̇ O2max significantly increased Tc after resting 30 min in a hot environment and remained significantly higher throughout exercise.(4) The mechanism for Tc increase is attributed to peripheral vasoconstriction,(4)

which prevents temperature dissipation. This mechanism is understandable considering

aspirin’s efficacy on the cardiovascular system, effectively reducing cardiac events (ie,

stroke and heart attack).(18) Compared to aspirin, naproxen has greater selectivity toward

COX 2, making naproxen highly effective at reducing pain and inflammation.(3) Unlike

aspirin, naproxen reversibly inhibits platelet aggregation,(1) making naproxen less

effective at reducing cardiac events. Because we did not measure skin blood flow we are

unable to make a definitive statement whether naproxen elicited any effect through

vasoconstriction, but based on our lack of Tc differences, we find it unlikely our naproxen

dosage caused changes in skin blood flow. With higher dosages (ie, prescription strength)

or longer use (ie, 7 days) the possibility exists that we would see similar responses as

Bruning et al.(4)

The significant increase in IL-6 pre- to post-exercise in our study was expected and similar to other moderate-intense cycling.(31) Like Tc, we found naproxen did not significantly affect IL-6. These results are consistent with other studies showing neither a

1000 mg aspirin (20) or 400 mg ibuprofen(27) dose significantly increased plasma

inflammatory cytokine concentrations. Aspirin is more selective toward COX 1 than 2,

meaning aspirin is less effective at relieving inflammation.(18) Considered equipotent

against COX 1 and 2, results on ibuprofen’s effects on exercise driven inflammation is

contradictory; though, dosage plays an important role. While 400 mg 2 hrs prior to ultra- distance running made no difference in IL-6(27), 600 mg the afternoon prior and 1200 mg

on the day of a ultra-distance race significantly increased post-race IL-6 compared to

controls.(17) The 3 220 mg naproxen doses over 24 hrs prior to exercise in our study did

not significantly increase or decrease IL-6 compared to placebos. If dosing was extended

across multiple days there may be significant effects.

Controlled laboratory exercise studies often do not measure NSAIDs, Tc, and

inflammation together, and even fewer field studies are available. Among a group of ultra-

marathon runners (n = 19), 6 participants reported using NSAIDs or other anti- inflammatory supplements (ie, fish oil).(10) Researchers found no correlation with NSAID use and IL-6. The researchers determined there was no effect on Tc; however, temperature was assessed aurally.(10) Aural temperature is known to be an inaccurate Tc assessment in exercising individuals.(5) The reported post-race temperatures (37.0 + 0.3°C)(10) may

not have accurately reflected individuals’ Tc when one considers a person’s normal

baseline Tc (37°C + 1°C)(16) and the environmental race conditions (30-40°C and 31-40%

RH).(10)

Individual Variability

Knowing a number of factors play a role in thermoregulation during exercise, we attempted to control nutrition, hydration, medication, physical activity, clothing, and illness. Furthermore, our study participants were non-heat acclimatized and moderately

endurance trained. Despite our attempts, a number of factors cannot be controlled and

explain some individual variability in existing EHS literature and in our results, particularly

when examining genders.

Though not statistically significant, the IL-6 increase in our male participants was greater than females. Also, these concentrations tended to stay elevated at 3 hrs post-

exercise. Lack of statistical significance likely comes from the large standard deviations

post-exercise among males in all conditions. Timmons et al.(31) compared immune

responses in males to females during different menstrual phases using and not using oral

contraception. Result indicated no significant differences in IL-6 levels from pre- to post- cycling.(31) Skeletal muscle contraction during exercise produces a high amount of IL-6,

which increases plasma IL-6.(30) Our male participants did not weigh significantly more

than females, but males had greater muscle mass, as indicated by their significantly lower

body fat %. The higher mean IL-6 and large variations in male participants could be caused

by muscle induced IL-6. Within the males, IL-6 means were lower post-exercise with

naproxen, suggesting there may have been some effects, but this did not continue at 3 hrs.

We identified some overall Tc differences. Both male and female Tc averaged

38.2°C immediately post-cycling, but males continued to increase during the 5 min cool

down, reaching 38.4°C and remaining significantly higher 10 min post-cycling. This

elevation may be explained by a higher metabolic heat production,(8) less body fat and slightly higher V̇ O2max. Female pre- and 3 hr post-exercise Tc was significantly higher than

males. This is likely due to the naturally higher Tc during the luteal phase (~ 0.3°C

higher).(23) We did not control for menstrual cycle, but we did not find significant

differences in resting Tc based on the individual’s phase (follicular or luteal). Oral

22 contraceptives increase Tc during moderate-intense exercise in the heat(15) similar to that seen in non-oral contraceptive users. In our one participant who did not use birth control, we found during the luteal phase, which happened to be when she completed NpxHeat, she experienced her highest post-exercise Tc and IL-6. This was not found during the luteal phase in any other female participant. Further, this individual was the one participant who reported a previous EHI and had the highest body fat % and lowest V̇ O2max among females.

Another interesting consideration for individual confounding risk factors is in our failed trials. A female participant attempting to complete the NpxHeat trial began experiencing hypotension and reported severe nausea. Researchers ceased her trial for safety reasons. Though her pre-trial measures were within normal limits, upon inquiry, the participant reported previous GI illness within a few days of the trial and feeling tired. One male participant was unable to begin a data collection trial due to extreme GI symptoms and illness after initiating the naproxen. The individual withdrew after a later trial due to the intensity of the study. These two examples support the need to use a multifaceted approach to recognize and monitor individuals at-risk for adverse events. Additionally, it is important to inquire about NSAID use in persons who may present with signs and symptoms before or during exercise.

Limitations and Future Research

The primary limitation to this study was using a constant HR during 80 min of cycling rather than using the work rate corresponding to 70% V̇ O2max. The purpose of a constant HR was to allow us to better determine the direct effects of naproxen on dependent measures. If work rate had been used we would expect an increased cardiovascular strain, particularly in the heat, which could increase Tc. However, by using a constant HR, the

cycling intensity during heat trials was inherently less than ambient due to cardiovascular

alterations (increased HR and BP) in response to thermal stress. With the available data, we are unable to identify specific work rate differences between heat and ambient trials during the 80 min. We acknowledge using constant HR likely limited us from finding significant differences between experimental conditions. Additionally, we could have

measured oxygen consumption to better control for work rate during each trial. Many

exercise bouts are completed at a set intensity rather than focusing on a target HR; however,

our results are useful for individuals who may use a set HR to pace themselves during

endurance exercise and then “go all-out” at the finish. Our second limitation is only

measuring IL-6, which is a prominent pro-inflammatory cytokine that increases early

during immune responses. Another prominent pro-inflammatory cytokine, tumor necrosis

factor-alpha increases several hours after exercise.(19) Examining this cytokine at 3 hrs

post-cycling and day-post would provide a better idea of naproxen’s effects. Third, diet

and physical activity logs indicated participants followed pre-data collection guidelines for

physical activity and nutrition. We assume participants were honest with following

guidelines and completing the logs.

Future research should examine naproxen and other non-aspirin NSAIDs in higher

doses, particularly looking at prescription strength and/or long-term use (ie, 7 days, 14

days, etc.). Research is warranted in hot environments using greater cycling intensity and

other exercise modes. Running is known to add strain on the GI tract,(24) which may

exacerbate responses. Field studies should be conducted in both long distance cycling and

running, as well as field sports where there is alternating high intensity activity and rest

periods. Considering GI barrier integrity and inflammation driven SIR closely resembles

24 models for EHS, research should examine plasma endotoxin concentration and GI permeability related to NSAID use and exercise. We also suggest additional research on potential gender differences. Examining females using and not using oral contraception is warranted as well as during different menstrual phases. Studies in individuals with musculoskeletal injuries or conditions potentially compromising the immune system are pertinent. Finally, future research is needed regarding confounding risk factors, such as sleep deprivation, nutrition, and especially hypohydration. Hypohydration places additional strain on the cardiovascular and thermoregulatory systems, which, if combined with NSAID effects, could promote Tc increase.

Conclusion

Our results suggest hydrated males and females completing moderate-intense exercise in either ambient or hot environments taking a 24 hr naproxen dose do not experience increased Tc or IL-6. These results are important for both physically active individuals and those working with persons who may be taking naproxen. While we did not find negative impacts on thermoregulation or inflammation, we encourage individuals to be aware of the potential adverse effects that NSAID may have on the GI tract that could perpetuate thermal intolerance during exercise. This understanding is especially vital in individuals with other confounding risk factors (eg, previous illness, sleep deprivation, repeated intense exercise bouts, hypohydration, etc.).

REFERENCES

1. Naproxen Sodium. National Center for Biotechnology Information. https://pubchem.ncbi.nlm.nih.gov/compound/23681059. Accessed March 13, 2016.

2. Sodium Salicylate. National Center for Biotechnology Information. https://pubchem.ncbi.nlm.nih.gov/compound/16760658. Accessed March 13, 2016.

3. Botting RM. Inhibitors of cyclooxygenases: mechanisms, selectivity and uses. J Physiol Pharmacol. 2006;57:113-24.

4. Bruning RS, Dahmus JD, Kenney WL, Alexander LM. Aspirin and clopidogrel alter core temperature and skin blood flow during heat stress. Med Sci Sports Exerc. 2013;45(4):674-82.

5. Casa DJ, Becker SM, Ganio MS, et al. Validity of devices that assess body temperature during outdoor exercise in the heat. J Athl Train. 2007;42(3):333-42.

6. Darling RL, Romero JJ, Dial EJ, Akunda JK, Langenbach R, Lichtenberger LM. The effects of aspirin on gastric mucosal integrity, surface hydrophobicity, and prostaglandin metabolism in cyclooxygenase knockout mice. Gastroenterology. 2004;127(1):94-104.

7. Downey JA, Darling RC. Effect of salicylates on elevation of body temperature during exercise. J Appl Phys. 1962;17(2):323-5.

8. Gagnon D, Kenny GP. Does sex have an independent effect on thermoeffector responses during exercise in the heat? J Physiol. 2012;590(23):5963-73.

9. Gill SK, Allerton DM, Ansley-Robson P, Hemmings K, Cox M, Costa RJ. Does short-term high dose probiotic supplementation containing lactobacillus casei attenuate exertional-heat stress induced endotoxaemia and cytokinaemia? Int J Sport Nutr Exerc Metab. 2015.

10. Gill SK, Teixeira A, Rama L, et al. Circulatory endotoxin concentration and cytokine profile in response to exertional-heat stress during a multi-stage ultra-marathon competition. Exerc Immunol Rev. 2015;21:114-28.

11. Jacobson ED, Bass DE. Effects of sodium salicylate on physiological responses to work in heat. J Appl Phys. 1964;19(1):33-6.

12. Lambert GP, Boylan M, Laventure JP, Bull A, Lanspa S. Effect of aspirin and ibuprofen on GI permeability during exercise. Int J Sports Med. 2007;28(9):722-6.

13. Lambert GP, Broussard LJ, Mason BL, Mauermann WJ, Gisolfi CV. Gastrointestinal permeability during exercise: effects of aspirin and energy-containing beverages. J Appl Physiol. 2001;90(6):2075-80.

14. Laporte J-R, Ibanez L, Vidal X, Vendrell L, Leone R. Upper gastrointestinal bleeding associated with the use of NSAIDs: newer versus older agents. Drug Saf. 2004;27(6):411-20.

15. Martin JG, Buono MJ. Oral contraceptives elevate core temperature and heart rate during exercise in the heat. Clin Physiol. 1997;17:401-8.

16. McArdle WD, Katch FI, Katch VL. Exercise Physiology: Energy, Nutrition, and Human Performance. Fifth ed. Baltimore, MD: Lippincott Williams & Wilkins; 2001.

17. Nieman DC, Henson DA, Dumke CL, et al. Ibuprofen use, endotoxemia, inflammation, and plasma cytokines during ultramarathon competition. Brain Behav Immun. 2006;20(6):578-84.

18. Patrono C, Garcia Rodriguez LA, Landolfi R, Baigent C. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med. 2005;353:2373-83.

19. Pedersen BK, Toft AD. Effects of exercise on lymphocytes and cytokines. Br J Sports Med. 2000;34:246-51.

20. Pernerstorfer T, Schmid R, Bieglmayer C, Eichler H-G, Kapiotis S, Jilma B. Acetaminophen has greater antipyretic efficacy than aspirin in endotoxemia: a randomized, double-blind, placebo-controlled trial. Clin Pharmacol Ther. 1999;66:51-7.

21. Pescatello LS, ed ACSM's Guidelines for Exercise Testing and Prescription. 9th ed. Baltimore, MD: Lippincott Williams & Wilkins; 2014. Arena R, ed. Exercise Testing.

22. Peters HPF, Wiersma WC, Akkermans LM, et al. Gastrointestinal mucosal integrity after prolonged exercise with fluid supplementation. Med Sci Sport Exerc. 2000;32(1):134-42.

23. Pivarnik JM, Marichal CJ, Spillman T, Morrow Jr. JR. Menstrual cycle phase affects temperature regulation during endurance exercise. J Appl Phys. 1992;72(2):543-8.

24. Rehrer NJ, Meijer GA. Biomechanical vibration of the abdominal region during running and bicycling. J Sports Med Phys Fitness. 1991;31(2):231-4.

25. Ryan AJ, Chang R-T, Gisolfi CV. Gastrointestinal permeability following aspirin intake and prolonged running. Med Sci Sport Exerc. 1996;28(6):698-705.

26. Sawka MN, Burke LM, Eichner ER, Maughan RJ, Montain SJ, Stachenfeld NS. American College of Sports Medicine position stand. Exercise and fluid replacement. Med Sci Sport Exerc. 2007;39(2):377-90.

27. Sepehri G, Pourranjbar M, Moshtaghi-Kashanian G-R, Khachaki AS, Sepehri E. The effect of a prophylactic dose of ibuprofen on plasma level of interleukin 1, interleukin 6 and tumor necrosis factor-alpha in a 1500 m running practice. Am J Appl Sci. 2011;8(1):50-4.

28. Spinas GA, Bloesch D, Keller U, Zimmerli W, Cammisuli S. Pretreatment with ibuprofen augments circulating tumor necrosis factor-a, interleukin-6, and elastase during acute endotoxinemia. J Infect Dis. 1991;163(1):89-95.

29. Starkie RL, Hargreaves M, Rolland J, Febbraio MA. Heat stress, cytokines, and the immune response to exercise. Brain Behav Immun. 2005;19(5):404-12.

30. Steensberg A, van Hall G, Osada T, Sacchetti M, Saltin B, Pedersen BK. Production of interleukin-6 in contracting human skeletal muscles can account for the exercise- induced increase in plasma interleukin-6. J Physiol. 2000;529(1):237-42.

31. Timmons BW, Hamadeh MJ, Devries MC, Tarnopolsky MA. Influence of gender, menstrual phase, and oral contraceptive use on immunological changes in response to prolonged cycling. J Appl Physiol (1985). 2005;99(3):979-85.

32. Wallace JL. Prostaglandins, NSAIDs, and gastric mucosal protection: why doesn't the stomach digest itself? Physiol Rev. 2008;88:1547-65.

Table 2.1. Participant Demographics (M + SD) Aggregate Male Female (N = 11) (N = 6) (N = 5) Age (yrs) 27.8 + 5.7 28.7 + 5.3 26.8 + 6.5 Weight (kg) 79.1 + 17.9 88.4 + 14.0 67.9 + 16.5 Height (cm) 177.0 + 9.5 183.2 + 5.3 169.5 + 7.8a Body fat (%) 15.2 + 8.3 10.7 + 4.0 20.6 + 9.1a

V̇ O2max (mL/kg) 41.4 + 5.7 43.6 + 5.3 38.7 + 5.3 aSignificantly different between genders (P < 0.04)

38.8 Control 38.6 Npx 38.4 Heat NpxHeat 38.2 ° C) 38.0

37.8

37.6 Core Temperature ( Core Temperature 30 37.4

37.2

37.0

36.8

36.6 Pre 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 5 10 15 30 45 60 75 90 105

Cycling (min) Rest (min) Figure 2.1. Mean Core Temperature during Cycling and Rest for Experimental Conditions Significant change in Tc during exercise (F1.7,67.3 = 150.5, P < 0.001) and rest (F2.0,81.5 = 201.6, P < 0.001)

a) Control

39.0

38.7

38.4 ° C) 38.1

37.8

37.5 Core Temperature ( Core Temperature

31 37.2

36.9

36.6

36.3

36.0 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 5 Cycing Time (min)

Figure 2.2.a. Regression and Participant Core Temperature during Cycling. Bold line represents mean Tc for condition. Shaded area indicates 10 min cycling at maximum effort. Final 5 min is an active cool down. Cubic regression equation: ŷ = 0.03x + 37.3; r2 = 0.59; 95% CI = 37.1, 37.5; P = 0.001.

b) Naproxen

39.0

38.7

38.4 ° C) 38.1

37.8

37.5 Core Temperature ( Core Temperature

32 37.2

36.9

36.6

36.3

36.0 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 5 Cycling Time (min)

Figure 2.2.b. Regression and Participant Core Temperature during Cycling. Bold line represents mean Tc for condition. Shaded area indicates 10 min cycling at maximum effort. Final 5 min is an active cool down. Cubic regression equation: ŷ = 0.02x + 37.4; r2 = 0.58; 95% CI =37.2, 37.6; P = 0.002.

c) Heat 39.0

38.7

38.4 ° C) 38.1

37.8

37.5

Core Temperature ( Core Temperature 37.2 33

36.9

36.6

36.3

36.0 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 5 Cycling Time (min)

Figure 2.2.c. Regression and Participant Core Temperature during Cycling. Bold line represents mean Tc for condition. Shaded area indicates 10 min cycling at maximum effort. Final 5 min is an active cool down. Linear regression equation: ŷ = 0.01x + 37.4; r2 = 0.54; 95% CI =37.2, 37.6; P < 0.001.

d) Heat and Naproxen 39.0

38.7

38.4 ° C) 38.1

37.8

37.5

Core Temperature ( Core Temperature 37.2 34

36.9

36.6

36.3

36.0 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 5 Cycling Time (min)

Figure 2.2.d. Regression and Participant Core Temperature during Cycling. Bold line represents mean Tc for condition. Shaded area indicates 10 min cycling at maximum effort. Final 5 min is an active cool down. Linear regression equation: ŷ = 0.01x + 37.4; r2 = 0.56; 95% CI =37.2, 37.6; P < 0.001.

4.4 Control a a a Npx 4.0 Heat 3.6 a NpxHeat

3.2

2.8

b 2.4 b

2.0 a,b 35 6 (pg/ml) 6

- a,b

IL 1.6

1.2

0.8

0.4

0.0 Pre Post 3 hr Post

Figure 2.3. Mean IL-6 Pre-, Post-, and 3 Hrs Post-Cycling for Experimental Conditions a N = 7. Significant change over time (F2, 48 = 41.8, P < 0.001). Significantly higher than pre- within condition (P < 0.04). bSignificantly lower than post- within condition (P < 0.04).

CHAPTER 3

NAPROXEN EFFECTS ON HYDRATION AND ELECTROLYTE MEASURES DURING CYCLING IN

THE HEAT1

1Dawn M. Emerson, Toni M. Torres-McGehee, Stephen CL Chen, J. Larry Durstine, J. Mark Davis, Craig E. Pfeifer, Charles C. Emerson, Justin V. Stone, Joseph D. Bivona. To be submitted to J Athl Train.

ABSTRACT

Context: Non-steroidal anti-inflammatory drugs (NSAIDs) can cause a number of adverse

effects, including impaired fluid-electrolyte balance. There is a lack of controlled

laboratory studies examining NSAID effects on fluid and electrolyte regulation during

exercise.

Objective: To examine the effects of naproxen, a commonly used NSAID, on hydration

and electrolyte measures in hydrated humans cycling in a hot environment.

Design: Double-blind, randomized and counterbalanced, cross-over design.

Setting: Laboratory.

Patients or Other Participants: A total of 11 moderately endurance trained volunteers (6 male and 5 female; age = 27.8 + 6.5 yrs, weight = 79.1 + 17.9 kg, height = 177 + 9.5 cm,

V̇ O2max = 41.4 + 5.7 mL/kg) completed this study.

Intervention(s): Participants were randomly assigned to complete 4 trials: 1) placebo and ambient (Control); 2) placebo and heat (Heat); 3) naproxen and ambient (Npx); and 4) naproxen and heat (NpxHeat).

Main Outcome Measure(s): Hydration (plasma osmolality, urine osmolality, urine specific gravity, and body mass change), electrolyte (plasma sodium and plasma potassium concentration), and cardiovascular (heart rate and blood pressures) measures were taken pre-, post- and 3 hrs post-90 min cycling. We also measured fluid volume (Fvol) and urine volume (Uvol).

Results: We found no statistically significant differences between experimental conditions for hydration, electrolyte, or cardiovascular measures. All participants began hydrated and became more hydrated throughout trials. Mean aggregate plasma sodium was < 135

mmol/L at pre-, post-, and 3 hrs post-exercise, but did not significantly decrease over time.

Overall plasma potassium significantly increased pre- to post-exercise (P = 0.02). Though not statistically significant, mean Fvol was greater and Uvol lower during naproxen trials compared to placebos.

Conclusion: A 24 hr naproxen dose did not significantly alter hydration or electrolyte measures during 90 min cycling in either ambient or hot conditions. The trend for naproxen to increase Fvol and decrease Uvol suggests the start of fluid retention, which should be a

concern for individuals at risk for hyponatremia or with pre-existing cardiovascular conditions (ie, hypertension).

Key Words: NSAID, urine specific gravity, plasma osmolality, plasma sodium, urine osmolality

INTRODUCTION

Non-steroidal anti-inflammatory drugs (NSAIDs) include a variety of prescription and

non-prescription medications including aspirin, ibuprofen, indomethacin, and naproxen.

An individual drug’s effectiveness and side effects are dependent on its chemical makeup

and selectivity toward inhibiting cyclooxygenase (COX). In general, all NSAIDs work to

relieve pain and inflammation by preventing COX from catalyzing the conversion of

arachidonic acid (a fatty acid released from cell membranes during injury/inflammation)

to prostaglandin (PG).1 There are numerous PG subsets that are responsible for not only

promoting pain and inflammation, but also maintaining blood pressure (BP), plasma

volume, and renal function. Although less imperative in normal healthy individuals, PGs

are critical in maintaining homeostasis during altered cardiovascular states (eg,

hypotension) or altered fluid regulation (eg, hypovolemia and sodium imbalance)2,3 –

common conditions experienced during physical activity. Inhibiting PGs can perpetuate

altered cardiovascular and renal function, potentially leading to serious fluid and

electrolyte imbalances (eg, hyponatremia, edema formation, and hypertension) or even

acute renal failure.3,4

Existing literature evaluating the relationship between NSAIDs and fluid regulation

during physical activity is primarily centered on observational studies examining exertional

hyponatremia (defined as a plasma sodium concentration [P[Na+]] < 135 mmol/L).4-6 The

mechanism for NSAIDs to promote hyponatremia is due to their ability to decrease urine

production and increase water retention, which dilutes P[Na+].7 To our knowledge, the only controlled study examining this relationship is by Dumke et al.,8 who found 600 mg of

ibuprofen pre- and 1200 mg during an ultra-distance marathon did not significantly alter

plasma electrolytes. Controlled laboratory studies examining NSAID effects on fluid-

electrolyte balance during exercise is lacking. Thus, as part of a larger study, we sought to

determine acute dose effects of a commonly used over the counter NSAID, naproxen, on

fluid and electrolyte measures during moderate-intense endurance exercise in a hot or ambient environment. We hypothesized naproxen would increase hydration measures, indicating further hydration (ie, euhydration or hyperhydration) compared to placebo controls. We also hypothesized naproxen would significantly decrease P[Na+] compared to placebo controls.

METHODS

Design

We used a double-blind, randomized and counterbalanced, cross-over design. The

independent variables were placebo or naproxen and hot or ambient environment. The

dependent variables were: plasma osmolality (Posm), urine osmolality (Uosm), urine

specific gravity (Usg), percent change in body mass (%BM), P[Na+], plasma potassium

concentration (P[K+]), fluid volume consumed (Fvol), urine volume (Uvol), heart rate

(HR), and blood pressure (BP).

Participants

Eleven participants (5 male, 6 female) were recruited from the local community.

Participants read and signed an Institutional Review Board approved informed consent form (Appendix A) prior to participation. Potential participants completed a health and injury history questionnaire (Appendix B) and were accepted if he/she was free of

cardiovascular, respiratory, and metabolic disorders; musculoskeletal disorders preventing

cycling exercise; fluid and electrolyte balance disorders; and gastrointestinal and

swallowing disorders. Current prescription and non-prescription medication use was also

asked. Females were asked supplemental questions on menstrual cycle and birth control.

Potential participants then completed a graded cycling V̇ O2max test (Appendix E.2) to

determine qualification as moderately trained (male V̇ O2max between 35-40 mL/kg; female

9 V̇ O2max between 32-40 mL/kg).

Experimental Conditions

Participants completed 4 experimental conditions: 1) placebo and ambient

(Control); 2) placebo and heat (Heat); 3) naproxen and ambient (Npx); and 4) naproxen

and heat (NpxHeat). A minimum of 7 days separated each trial. A 24 hr dose (3 capsules)

of placebo (cellulose) or naproxen (220 mg naproxen sodium/dose) was given to

participants with instructions to take 1 capsule at 16 hrs, 8 hrs, and 0 hrs prior to data

collection. Participants were given specific instructions to follow 24 hrs prior to data

collection, including directions for taking each capsule according to the manufacturer’s (ie,

naproxen sodium) directions. Participants then completed a 90 min cycling bout in either

a hot (mean temperature = 35.7 + 1.3°C, 53.2 + 3.2 % relative humidity [RH]) or ambient

(mean temperature = 22.7 + 1.8°C, 52.4 + 5.5% RH) environment. After exercise,

participants rested 3 hrs in an ambient environment (23°C, 56% RH).

Instruments and Protocols

Anthropometric Measures. Weight and body fat percentage was measured using a bioelectrical impedance analysis scale (Tanita SC-331S Body Composition Monitor,

Tanita Co., Tokyo, Japan). Height was self-reported.

Health and Injury History Questionnaire (Appendix B). The Health and Injury

History Questionnaire uses the Canadian Society for Exercise Physiology, which

developed the PAR-Q to identify individuals who should be seen by a medical doctor

before becoming more physically active and both the American Medical Society for Sports

Medicine and the American Orthopedic Society for Sports Medicine. These two

organizations released pre-participation physical exam forms used to identify individuals

at risk for injuries. Supplemental questions were added to identify individuals with

potential medical illness or injury (eg, gastrointestinal disorders, exertional heat illness

history, metabolic disorders, and daily medication use) that would exclude them from study

participation.

Cycle Exercise Protocol. Participants completed a 90 min cycling protocol on a

stationary bike (Monark Ergomedic 828E, Monark Exercise AB, Vansbro, Sweden). Prior

to a 3 min warm up, participants were provided their target HR, equivalent to 70%

10 V̇ O2max, to maintain during 80 min of steady state cycling. Participants were instructed to

reach the HR by the end of the 3 min warm up. At the end of 80 min, participants performed

10 min at maximum effort, with each participant given verbal encouragement by research assistants. The protocol concluded with a 5 min cool down.

Hydration and Electrolyte Measures. Participants were required to be euhydrated

prior to beginning data collection. Immediate hydration status was assessed using Usg.

Further hydration analysis was conducted using Posm, Uosm, and %BM. Euhydration was

defined as Posm < 290 mOsmols, Uosm < 700 mOsmols, Usg < 1.020, and %BM < 1%.11

We collected blood from an antecubital vein into 6ml lithium heparin vacutainer

tubes at pre-, post-, and 3 hrs post-exercise. Tubes were inverted several times to mix and immediately placed on ice. Samples were centrifuged at 3000 rpm for 15 min. Plasma was pipetted into microtubes and stored at -20°C until analysis. We measured osmolality using freeze point depression (Multi-sample Osmometer model 2020, Advanced Instruments,

Norwood, MA). To determine changes in P[Na+] and P[K+] we utilized ion-selective

electrodes (EasyLyte® Na/K electrolyte analyzer model REF 2277, Medica, Bedford, MA).

Normal P[Na+] was defined as > 135 mmol/L4 and normal P[K+] < 5 mmol/L.12

Urine was obtained pre-, post-, and 3 hrs post-exercise. A clean catch method was used in which the participant was instructed to urinate a small amount before placing the specimen cup in midstream to collect a minimum of 1 ounce of urine. We used a handheld clinical refractometer (model REF 312, Atago Company Ltd., Tokyo, Japan) to measure

Usg. Urine aliquots were stored in microtubes at -20°C until analysis for osmolality using freeze point depression.

Pre-, post-, and 3 hrs post-exercise BM was measured using a body composition analyzer (Tanita SC-331S Body Composition Monitor, Tanita Co., Tokyo, Japan).

Participants dressed in shorts and a t-shirt and voided urine before stepping on the scale.

Immediately post-exercise participants were asked to towel off sweat before weighing.

Participants were then allowed to change clothes and instructed to return with their “wet”

clothes to be weighed. Differences in “wet” versus “dry” clothes’ weight (measured at

another time) were used to adjust BM for sweat loss.

Urine Volume. All urine produced after pre-BM and hydration measures was collected into Uvol containers and urine cups (for Usg and Uosm measures). Total Uvol produced over the trial was measured using a graduated cylinder.

Fluid Volume. To maintain hydration during exercise, participants were instructed to drink 3.5 ml/kg of water every 15 minutes. Participants were allowed to consume more than the required Fvol if they desired. At the end of exercise, researchers measured total

Fvol consumed. During the 3 hr rest period, participants consumed water ad libitum and researches measured Fvol consumed during rest.

Cardiovascular. To ensure participants remained at safe limits during exercise,

HR was continuously monitored using Polar HR monitors (Polar Electro Inc., Lake

Success, NY). To determine effects from naproxen, pre-, post-, and 3 hr post-exercise HR and BP were assessed.

Diet and Activity Logs. To control for pre-data collection dietary and physical activity effects, participants were asked to track diet and physical activity for 3 days prior and 1 day after data collection using an online nutrition software FoodProdigyTM (ESHA

Research, Salem, OR). Participants were asked to mimic diet and physical activity habits

the 24 hrs prior to each trial.

Experimental Procedures (Figure 3.1)

Information Session. After consenting participants were determined free of

disqualifying medical conditions and to be moderately trained, a research assistant

randomly assigned an experimental condition order. Participants and primary investigators

were blind to whether participants were completing a naproxen or placebo trial.

Participants were familiarized with the online diet and activity log and the V̇ O2max test was used to familiarize participants to the stationary cycle. Participants were instructed to not take any analgesic or anti-inflammatory medications during the course of the study.

Pre-Data Collection. Seventy-two hrs prior to data collection, participants were

sent an email with instructions for completing the diet and physical activity log.

Participants were instructed to refrain from intense, vigorous exercise for 48 hrs.13 Twenty-

four hours prior, participants were provided written and verbal instructions for taking 2

capsules (placebo or naproxen), to consume a small meal prior to reporting to the

laboratory, encouraged to consume fluids to promote euhydration, refrain from all physical

activity, and attempt to sleep a “normal” amount.

Data Collection Session. Upon arrival to the laboratory, participants were verbally

asked if they took their 2 pills before taking the 3rd pill. Participants were provided a HR

strap and baseline HR and BP were assessed. Participants provided a urine sample and

baseline Usg was measured to ensure euhydrated prior to BM and blood measures.

Immediately following the cycling protocol participants weighed, provided a urine

sample and had post- blood collected. Participants then rested in a semi-reclined/seated

position where they were allowed to consume water ad libitum and given a snack. Snacks

were based on BM and the same for each trial. At the conclusion of the 3 hr rest period,

blood, urine, weight, BP, and HR were measured.

Statistical Analysis

IBM SPSS Statistics (version XXII; IBM Corporation, Armonk, NY) was used for

all analyses. Descriptive statistics (mean and standard deviations) for all dependent

variables were calculated. A one-way ANOVA was used to determine differences in

demographics between genders (eg, age, height, weight). A 3 (time) x 4 (condition) repeated measures ANOVA determined differences for plasma, urine, %BM, HR, and BP.

Greenhouse-Geisser corrections were used for P[Na+], BP, and HR because these variables

violated sphericity. Post hoc analysis was conducted for significant main effects using

Bonferroni corrections. We used a one-way ANOVA to determine differences between conditions and gender for Fvol consumed during exercise, total Fvol, and Uvol. To control

for body mass, Fvol was corrected to ml/kg and a one-way ANOVA determined differences

between conditions and gender. We used paired sample t-tests to determine differences in sweat rate and Fvol during exercise. Using G*Power (version 3.1.9.2, Heinrich Heine

University, Dusseldorf, Germany),14 post hoc power calculation with means and variances

for Usg and Uosm indicated a statistical power > 0.9. Significance level was set at P < 0.05

for all analyses.

RESULTS

We began the study with 16 participants; 2 participants dropped due to the study

intensity and 3 dropped due to the time commitment. Our final sample size = 11. There

was no significant difference between genders for age (mean = 27.8 + 5.7 yrs), weight (79.1

+ 17.9 kg), V̇ O2max (41.4 + 5.7 mL/kg), sweat rate (0.9 + 0.3 L), baseline resting HR (52.6

+ 6.6 bpm) and baseline resting BP (116/80 mmHg). Due to lost samples, the sample size

for Posm, P[Na+], and P[K+] ranged between conditions (Control = 5, Npx = 3-4, Heat =

4, and NpxHeat = 3-4). Diet logs indicated no significant differences between calorie, fat,

carbohydrate, sodium, and protein intake between conditions. On average, male daily

sodium intake was significantly greater than females (2.7 + 0.3 g vs. 2.0 + 0.2 g, P = 0.038;

Appendix F, Table F.2). Physical activity level 24 hrs prior to data collection was not significantly different between conditions and remained low to sedentary.

Cardiovascular

There was a significant main effect for HR over time (mean pre = 66.8 + 14.3 bpm,

post = 177.4 + 16.2 bpm, and 3 hr post = 65.5 + 11.8 bpm, P < 0.001). There was no

significant HR difference between conditions or genders. Similarly, a significant main

effect occurred for BP over time (mean pre = 118/73 mmHg, post = 137/72 mmHg and 3

hrs = 116/77 mmHg, P < 0.008). There were no significant differences between conditions.

We found a significant main effect for systolic BP between genders (P = 0.005). Post-hoc

analysis showed males were significantly higher at pre-, post- and 3 hrs post-exercise (P <

0.016). Regarding experimental conditions (Appendix F, Figure F.6), male systolic BP was significantly higher than females post-exercise in Npx (150.8 vs 124.4 mmHg, P = 0.017) and NpxHeat (144.2 vs 124.0 mmHg, P = 0.043). At 3 hrs post-exercise, males were significantly higher within Heat (121.5 vs 111.6 mmHg, P = 0.028).

Hydration

Hydration, Fvol, and Uvol for each experimental condition are presented in Table

3.1. All participants began trials euhydrated. Overall, Posm, Uosm, and Usg significantly

decreased pre- to 3 hrs post-exercise, indicating participants became more hydrated

throughout trials. All conditions maintained an average BM loss < 1%. No significant

differences were found in hydration measures between conditions at any time point.

Though not significantly different, mean Uvol was lower in naproxen trials versus placebos and during heat trials versus ambient (Table 3.1). There was no significant difference in

Uvol between genders (Appendix F, Table F.1).

Neither Fvol during exercise or total was significantly different between conditions.

As expected, participants tended to consume more fluid during exercise in hot conditions

versus ambient (Table 3.1). Mean Fvol during exercise and overall was higher in both

naproxen trials compared to placebos. This trend continued when corrected for body mass.

Participants’ mean Fvol during exercise (1.5 + 0.7 L) was significantly greater than their

sweat rate (0.9 + 0.3 L, P < 0.013). Males consumed significantly greater Fvol during

exercise and total than females (Appendix F, Table F.1). During NpxHeat males consumed significantly more during exercise than females (2.0 + 0.6 vs. 1.3 + 0.5 L, P = 0.046). When

corrected for BM, Fvol in males overall and during NpxHeat was not significantly different

than females. During the Npx condition overall Fvol approached significance (males = 1.9

+ 1.0 L and females = 0.9 + 0.5 L, P = 0.055) and trended to be different during exercise

with BM corrections (males = 20.7 + 8.8 ml/kg and females = 12.1 + 5.0, P = 0.087).

Electrolytes

Table 3.2 shows plasma electrolytes by experimental condition. Mean aggregate

P[Na+] was < 135 mmol/L at pre-, post-, and 3 hrs post-exercise. We found no statistically significant differences in P[Na+] between conditions at any time. Mean P[Na+] was highest

during NpxHeat. There was a significant main effect for P[K+] changes over time (P =

0.02), increasing, expectedly, pre- to post-exercise and decreasing after 3 hrs. There were

no significant differences between trials; however, both naproxen groups averaged higher than placebos post-exercise. Due to the low sample size, we were unable to determine any differences between genders.

DISCUSSION

We sought to determine whether naproxen adversely affected hydration and electrolyte measures during moderate-intense endurance cycling in either hot or ambient conditions. We based our hypotheses on previous research7 associating NSAIDs with promoting fluid retention through decreased Uvol and renal vasoconstriction, which dilutes electrolytes and increases blood pressure. Our results did not support our hypotheses, finding naproxen did not significantly decrease P[Na+], Posm, urine hydration measures or increase cardiovascular strain compared to placebos. We also found responses did not differ between either hot or ambient environmental conditions.

Fluid and Electrolyte Balance

Naproxen is highly effective at inhibiting renal PGs, resulting in decreased renal blood flow.3 Naproxen also suppresses the renin-angiotensin-aldosterone system, which is responsible for maintaining BP, plasma volume, and electrolyte balance.7 Specific renal consequences include decreased glomerular filtration rate, increased vasopressin (anti- diuretic hormone), increased sodium retention, and decreased Uvol. Independently or concurrently, these effects cause water retention and vasoconstriction.7 In extreme cases, cell necrosis, interstitial nephritis, and renal failure may develop.3 Though these conditions can become severe, milder consequences include electrolyte imbalance, edema, and hypertension.4,7

Decreasing Uvol and increasing water retention is what makes NSAIDs a suspected

contributor to hyponatremia. Despite this, few studies have examined this relationship,

with most being observational studies with conflicting results.5,6,15,16 Ultra-distance triathletes using NSAIDs had significantly lower post-race P[Na+] compared to non-

NSAID users (mean = 140.2 mmol/L and 141.1 mmol/L, respectively, P < 0.02).5 The 6

participants who experienced hyponatremia all reported using NSAIDs.5 In 15 marathon runners hospitalized with severe hyponatremia, 28.6% used NSAIDs.15 However, other

observational studies found no relationship to hyponatremia.6,16,17 One inherent issue with

existing literature is not knowing what type and how much of an NSAID someone took.

All NSAIDs inhibit PGs, but their COX selectively and chemical make-up, along with an

individual’s unique response, means each NSAID exerts effects differently.

Another contributing factor for developing hyponatremia is an individual

consuming water (hypotonic fluid) in excess of fluid lost through sweat and respiration.4

If combined with other factors that dilute P[Na+], the individual can be at greater risk for hyponatremia. Current recommendations advise matching individual sweat losses rather than drinking in excess or following generalized protocols. Calculating sweat loss is relatively simple ([pre-activity body weight – post-activity body weight + fluid volume consumed – urine volume]/exercise time).18 Either lack of knowledge, ability, or resources

may prohibit the average person from calculating his/her sweat rate. Instead, the person

may guestimate or trial and error fluid intake during endurance activity. We chose to

provide a standardized water protocol (3.5 ml/kg), rather than match sweat rates, and

participants were allowed to consume more if desired. In part, using a standardized intake

mimicked what an individual who does not know their personal water needs may do. We

found consuming water volumes exceeding sweat loss during 90 min cycling did not

significantly deplete P[Na+] and there was no effect from NSAIDs. This is likely due to the

short exercise duration. Though exertional hyponatremia has occurred in football,19-21 it is

typically associated with long-duration endurance exercise (> 3 hrs).4,22 Our exercise

session likely did not allow enough time to significantly deplete P[Na+]. During the 3 hr

rest, when participants were allowed to drink water ad libitum they maintained euhydration

and plasma electrolyte balance.

One interesting note is our mean P[Na+] was < 135 mmol/L in all conditions and

time points except pre- and 3 hr post-exercise NpxHeat. Plasma sodium < 135 mmol/L

without signs or symptoms is considered biochemical or asymptomatic hyponatremia.22

Many individuals do not experience symptoms until P[Na+] decreases below 130 mmol/L

and severe hyponatremia typically occurs below 120 mmol/L.11 Our participants

maintained normal diets, consuming an average 2401.1 + 759.2 mg of sodium daily. The

low P[Na+] can be explained by participants being instructed and attempting to arrive to

the laboratory hydrated and by maintaining hydration during our exercise protocol. Pre-

exercise Usg, Uosm, and Posm indicated our participants were slightly hyperhydrated and

became extremely hyperhydrated23 by 3 hrs post-exercise.

Also interesting was that NpxHeat averaged more fluid consumed and had the lowest Uvol. Due to our low plasma sample size and lack of significance, we have difficulty

definitively explaining why this response occurred. The trend with naproxen to produce

less urine while consuming more fluid suggests participants were retaining water. Since

we did not see significant decreases in P[Na+], Posm, or Usg pre- to post-exercise, we presume a portion of water remained in the stomach. Intense exercise slows gastric

emptying, which prevents fluid from being absorbed and/or used to maintain physiological

processes.24

In theory, increased Fvol and decreased Uvol should lead to BM gains. We found

no significant difference in BM, which, again, may be attributed to exercise duration.

However, both the Npx and NpxHeat averaged pre- to post-exercise BM losses, while both

Control and Heat gained slightly or did not change (Table 3.1). Once exercise ceased (post- to 3 hrs post-exercise) this trend flipped, with Control and Heat indicating more weight loss than Npx and NpxHeat. Once exercise ceased, the possibility exists that GI and renal function returned to normal, promoting gastric emptying and water excretion as necessary to maintain fluid-electrolyte balance. Support for this explanation is seen with the slightly higher Uvol in Control and Heat. On the other hand, naproxen’s effects continuing throughout the rest period would explain the slight weight gain and lower average Posm at

3 hrs post-exercise. This thought is an important note considering reported cases of hyponatremia developing hours after activity.19,21 Sustained water retention combined with continued hydration would place the person at even greater risk for diluting P[Na+].

Regarding potassium, our participants remained within normal P[K+] levels (< 5

mmol/L).12 Potassium is tightly regulated by the kidneys, because elevated levels can cause cardiac arrhythmias and death.12 Even during exercise, when plasma levels can

quickly spike due to working muscles releasing or failing to re-uptake potassium,12 the

kidneys work efficiently to clear potassium. However, potassium will stay elevated if renal

blood flow is decreased25 or through decreased energy metabolism12 and increased oxygen

tension.26 NSAIDs can induce hyperkalemia,27 but there is little research regarding this

association during exercise. The increase in our overall P[K+] means are similar to those

reported in existing literature.5,8,28 While not significantly different between conditions, the

higher means with naproxen compared to placebos suggest there could be some effect from

the NSAID. Among triathletes, NSAIDs resulted in significantly higher post-race P[K+] compared to those not using NSAIDs (4.51 + 0.46 mmol/L and 4.35 + 0.43 mmol/L,

respectively, P = 0.002).5 In contrast, Dumke et al,5 found over the counter ibuprofen did

not significantly affect P[K+] in ultra-distance runners. Since type of NSAIDs were not reported, it is not possible to make comparisons among specific NSAIDs. Research has

established, compared to no NSAID exposure, odds for developing kyperkalemia with

ibuprofen are lower (odds ratio = 0.94) compared to naproxen (odds ratio = 1.08).27 Using

a higher naproxen dose for a longer duration may induce significant P[K+] changes during exercise.

Cardiovascular

Our results indicate 3 220 mg naproxen doses over 24 hrs did not significantly affect pre-, post-, or 3 hr post-exercise HR or BP. Cardiovascular responses often occur

concurrently with renal responses, since altering fluid-electrolyte balance can control

plasma volume and BP. Disregarding renal effects, naproxen is less effective at directly

altering the cardiovascular system because naproxen is more COX 2 selective.29 This

naproxen effect is in comparison to aspirin, which is more selective toward COX 1.30

Further, naproxen reversibly inhibits platelets while aspirin irreversibly inhibits platelet aggregation, making aspirin highly effective at reducing cardiovascular events (ie, stroke, heart attack).30 Lack of cardiovascular effects could be attributed to naproxen itself or to

our methodology. By maintaining hydration we limited cardiovascular changes that would

otherwise be seen with hypohydration. We also limited our participants to healthy, young,

moderately trained individuals. The same cardiovascular results may not be seen in

different populations (eg, older, poor physical conditioning, cardiovascular or renal

disease).

Gender

Previous literature identifies females are at higher risk for hyponatremia because of smaller body weights, excessive water intake, and longer race times compared to males.4

Though these factors play a role, hormonal differences between genders is also an

important consideration. Resting plasma vasopressin varies in females depending on

menstrual phase. Vasopressin is significantly lower than males during the early follicular phase, but not during luteal.31 These variances may help explain why we found differences

in Fvol between genders. Males consumed significantly more fluid than females both

during exercise and overall. Interesting, males consumed significantly more fluid during

exercise in the NpxHeat, and approached significance in Npx, compared to females.

Gender differences did not occur during Control and Heat and we found no difference in

females’ Fvol between conditions. Considering the change in vasopressin during menstrual

phases, which we did not account for, the lack of significant findings among females could

be partially explained by hormones.

Related to Fvol, thirst is listed as a potential side effect for several NSAIDs. There

is extremely limited research examining NSAIDs on thirst or Fvol during exercise. Among

NSAID and non-NSAID users completing an 82 km mountain run (n = 44; 36 males, 8

females), NSAID users (n = 16) consumed significantly more fluid than non-users (6.1 +

1.5 L vs 5.1 + 2.2 L, P = 0.08).17 Our results support, at least in males, naproxen increases

fluid intake. We used naproxen sodium, which is bioequivalent to naproxen except for rate

of absorption. The salt of a given NSAID is commonly used because it allows the drug to

dissolve faster and exert effects earlier. A 220 mg naproxen sodium dose contains

approximately 20 mg of sodium. Not all salts exert the same effects and 20 mg is relatively

low, but Wemple et al,32 found 46 mg/Lof sodium chloride added to a flavored beverage

significantly increased fluid intake. Naproxen sodium may stimulate thirst. Another

mechanism for thirst stimulation is NSAIDs increasing vasopressin.7 Vasopressin is

typically upregulated during dehydration in order to restore plasma volume, and its increase

is positively and linearly associated with thirst.31

Extensively examining cardiovascular effects between genders is difficult because

we cannot compare plasma measures and did not assess hormones. We identified no

differences between or within genders for HR. Blood pressure is generally higher for men

than females,33 which is attributed to physical differences, hormones, and vascular responsiveness.31 Male baseline BP was slightly higher than females, but not significantly.

Naproxen significantly elevated post-exercise male systolic BP compared to females. This did not occur during Control and Heat. The increased Fvol with naproxen in males could partially explain the higher systolic BP. Seemingly, males would have a higher plasma volume, which would increase BP.

Limitations and Future Research

One limitation to our study is not measuring aldosterone and vasopressin.

Measuring these hormones would provide a better explanation on how naproxen influences fluid and electrolyte regulation. Measuring plasma naproxen levels would have been useful

to determine the drug’s concentration at 3 hrs post-exercise. Naproxen has a long half-life

compared to other NSAIDs. Therefore, we would expect naproxen to continue exerting

effects during the 3 hr rest, while other NSAIDs would not. This knowledge would be

important when considering post-activity effects. Our 90 min cycling was likely too short

to elicit significant P[Na+] changes, considering much of the hyponatremia research is in

marathon and ultra-distance events. Due to lost samples, our sample size for plasma

electrolytes and osmolality was low, preventing us from achieving statistical significance between conditions. Finally, we did not control for menstrual phase.

Future research in laboratory settings is warranted to determine the relationship

between NSAID use and hydration-electrolyte balance during exercise. Because each

NSAID is unique, studies should evaluate different types, dosages, and length of use.

Research should evaluate longer endurance exercise bouts to elicit plasma electrolyte

depletion. Shorter exercise bouts are relevant, as well, considering the potential for

electrolyte depletion associated with other risk factors such as inadequate nutrition,

excessive hypotonic fluid intake, and/or excessive sweat sodium loss. Using different

hydration regimens (metered versus ad libitum) and fluid types (eg, carbohydrate-

electrolyte beverages) is also merited. Considering the trend for increased Fvol with

naproxen, evaluating vasopressin and thirst would be interesting. Along these lines,

examining the salt of different NSAID types would provide meaningful information.

Finally, controlled studies evaluating markers of renal function and gender differences,

including differences during menstrual phases is merited.

Conclusion

Acute naproxen intake did not adversely affect hydration, electrolyte, or

cardiovascular measures during cycling in either hot or ambient conditions. An important

note is the context of our findings, and that we identified some trends. Our participants

were hydrated, moderately endurance trained, free of many confounding risk factors, and the cycling protocol was moderate in duration (90 min). Second, NSAIDs vary in selectivity and chemical make-up. Our participants consumed the over the counter strength, recommended daily naproxen sodium dose.

We encourage clinicians and other personnel to consider an individual’s personal risk factors when recommending naproxen. Finding higher post-exercise systolic BP with naproxen in males versus females warrants consideration for potential adverse events during exercise in males with pre-exiting hypertension. Further, we found naproxen promoted greater fluid intake in males during ambient and hot environments. Beginning exercise slightly hyperhydrated and consuming water in excess of sweat rate during a 90 min exercise time began to show the trend for naproxen to induce water retention through decreased Uvol. Though our participants maintained electrolyte balance, their P[Na+] was lower than 135 mmol/L. Together, these results show the importance in considering individuals who may have risk factors for renal and/or cardiovascular imbalances during exercise.

REFERENCES

1. Straus DS, Glass CK. Cyclopentenone prostaglandins: new insights on biological activities and cellular targets. Med Res Rev. 2001;21(3):185-210.

2. Prostanoids - prostaglandins, prostacyclins, and thromboxanes. AOCS Lipid Library; 2014. Accessed 5/12/2015.

3. Garella S, Matarese RA. Renal effects of prostaglandins and clinical adverse effects of nonsteroidal anti-inflammatory agents. Medicine. 1984;63(3):165-181.

4. Rosner MH, Kirven J. Exercise-associated hyponatremia. Clin J Am Soc Nephrol. 2007;2(1):151-161.

5. Wharam PC, Speedy DB, Noakes TD, Thompson JM, Reid SA, Holtzhausen LM. NSAID use increases the risk of developing hyponatremia during an Ironman triathlon. Med Sci Sport Exerc. 2006;38(4):618-622.

6. Almond CS, Shin AY, Fortescue EB, et al. Hyponatremia among runners in the Boston Marathon. N Engl J Med. 2005;352(15):1550-1556.

7. Cheng HF, Harris RC. Renal effects of non-steroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors. Curr Pharm Des. 2005;11(14):1795-1804.

8. Dumke CL, Nieman DC, Oley K, Lind RH. Ibuprofen does not affect serum electrolyte concentrations after an ultradistance run. Br J Sports Med. 2007;41(8):492-496.

9. Pescatello LS, ed ACSM's Guidelines for Exercise Testing and Prescription. 9th ed. Baltimore, MD: Lippincott Williams & Wilkins; 2014. Arena R, ed. Exercise Testing.

10. Starkie RL, Hargreaves M, Rolland J, Febbraio MA. Heat stress, cytokines, and the immune response to exercise. Brain Behav Immun. 2005;19(5):404-412.

11. Sawka MN, Burke LM, Eichner ER, Maughan RJ, Montain SJ, Stachenfeld NS. American College of Sports Medicine position stand. Exercise and fluid replacement. Med Sci Sport Exerc. 2007;39(2):377-390.

12. Clausen T. Hormonal and pharmacological modification of plasma potassium homeostasis. Fundam Clin Pharmacol. 2010;24(5):595-605.

13. Peters HPF, Wiersma WC, Akkermans LM, et al. Gastrointestinal mucosal integrity after prolonged exercise with fluid supplementation. Med Sci Sport Exerc. 2000;32(1):134-142.

14. Faul F, Erdfelder E, Lang A-G, Buchner A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods. 2007;39(2):175-191.

15. Davis DP, Videen JS, Marino A, et al. Exercise-associated hyponatremia in marathon runners: a two year experience. J Emerg Med. 2001;21(1):47-57.

16. Hsieh M, Roth R, Davis DL, Larrabee H, Callaway CW. Hyponatremia in runners requiring on-site medical treatment at a single marathon. Med Sci Sport Exerc. 2002;34(2):185-189.

17. Scotney B, Reid S. Body weight, serum sodium levels, and renal function in an ultra- distance mountain run. Clin J Sport Med. 2015;25:341-346.

18. Casa DJ, Armstrong LE, Hillman SK, et al. National Athletic Trainers' Association Position Statement: fluid replacement for athletes. J Athl Train. 2000;35(2):212-224.

19. Stevens A. Update: Douglas County football player has died. The Atlanta Journal- Constitution. August 11, 2014.

20. Blevins R, Apel T. Doctor: Wilbanks' death unpreventable, freak occurrence. The Clarion-Ledger2014.

21. Creamer M, Hagedorn E. Family in disbelief that drinking too much water killed football player. The Bakersfield Californian2008.

22. Noakes TD, Sharwood K, Speedy D, et al. Three independent biological mechanisms cause exercise-associated hyponatremia: evidence from 2,135 weighed competitive athletic performances. Proc Natl Acad Sci U S A. 2005;102(51):18550-18555.

23. Armstrong LE, Pumerantz AC, Fiala KA, et al. Human hydration indices: acute and longitudinal reference values. Int J Sport Nutr Exerc Metab. 2010;20(2):145-153.

24. Robinson TA, Hawley JA, Palmer GS, et al. Water ingestion does not improve 1-h cycling performance in moderate ambient temperatures. Eur J Appl Physiol. 1995;71:153-160.

25. Franscesconi RP, Willis JS, Gaffin SL, Hubbard RW. On the trail of potassium in heat injury. Wilderness Environ Med. 1997;8:105-110.

26. Kilburn KH. Muscular origins of elevated plasma potassium during muscular exercise. J Appl Phys. 1966;21:675-678.

27. Lafrance JP, Miller DR. Dispensed selective and nonselective nonsteroidal anti- inflammatory drugs and the risk of moderate to severe hyperkalemia: a nested case- control study. Am J Kidney Dis. 2012;60(1):82-89.

28. McKenney MA, Miller KC, Deal JE, Garden-Robinson JA, Rhee YS. Plasma and electrolyte changes in exercising humans after ingestion of multiple boluses of pickle juice. J Athl Train. 2015;50(2):141-146.

29. Botting RM. Inhibitors of cyclooxygenases: mechanisms, selectivity and uses. J Physiol Pharmacol. 2006;57:113-124.

30. Patrono C, Garcia Rodriguez LA, Landolfi R, Baigent C. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med. 2005;353:2373-2383.

31. Wenner MM, Stachenfeld NS. Blood pressure and water regulation: understanding sex hormone effects within and between men and women. J Physiol. 2012;590(23):5949-5961.

32. Wemple RD, Morocco TS, Mack GW. Influence of sodium replacement on fluid ingestion following exercise-induced dehydration. Int J Sport Nutr. 1997;7:104-116.

33. Christou DD, Jones PP, Jordan J, Diedrich A, Robertson D, Seals DR. Women have lower tonic autonomic support of arterial blood pressure and less effective baroreflex buffering than men. Circulation. 2005;111(4):494-498.

Table 3.1. Hydration Measures, Fluid Volume, and Urine Volume for Experimental Conditions (M + SD)

Aggregate Placebo Npx Heat NpxHeat

Pre 286.6 + 6.4a 285.8 + 5.3 288.8 + 4.8 285.5 + 10.1 287.8 + 6.6 Posm Post 284.8 + 6.7 288.2 + 6.6 281.9 + 3.9 285.9 + 10.6 283.8 + 4.9 (mOsm/L) 3 hr post 282.4 + 5.9 286.4 + 3.3 277.4 + 6.6 283.6 + 6.9 281.1 + 4.9

Pre 399.5 + 172.1c 441.6 + 187.5 431.8 + 193.5 341.4 + 131.7 389.9 + 183.4 Uosm Post 277.3 + 236.0 246.8 + 223.7 244.2 + 184.1 233.1 + 213.9 390.2 + 310.0 (mOsm/L)b 3 hr post 176.2 + 132.9 192.4 + 107.8 185.6 + 130.4 201.3 + 173.0 122.7 + 113.3

Pre 1.012 + 0.005 1.013 + 0.005 1.014 + 0.006 1.011 + 0.004 1.012 + 0.004 Usg Post 1.011 + 0.008 1.010 + 0.007 1.010 + 0.008 1.010 + 0.008 1.013 + 0.009 3 hr post 1.007 + 0.006d 1.007 + 0.005 1.007 + 0.006 1.007 + 0.006 1.007 + 0.007

59 Pre - Post -0.1 + 0.8 0.1 + 0.8 -0.3 + 1.1 0.0 + 0.6 -0.1 + 0.8

∆BM (%) Pre - 3 hr -0.4 + 0.9 -0.6 + 0.4 -0.1 + 0.6 -0.5 + 0.6 -0.5 + 0.9 Post - 3 hr -0.3 + 0.7 -0.5 + 0.8 0.0 + 1.3 -0.4 + 0.5 -0.0 + 0.6

Uvol (ml) 1012.9 + 519.1 1157.3 + 681.4 1075.3 + 572.3 982.6 + 508.6 836.6 + 214.2

Exercise 1.5 + 0.7 1.1 + 0.4 1.4 + 0.9 1.6 + 0.6 1.7 + 0.7 Fvol (L) Total 2.1 + 1.1 1.7 + 0.6 2.1 + 1.5 2.2 + 1.0 2.5 + 1.3

Exercise 17.8 + 6.3 14.2 + 4.2 16.8 + 8.3 19.6 + 4.9 20.4 + 5.8 Fvol (ml/kg) Total 25.9 + 10.0 21.5 + 6.6 25.5 + 13.4 27.3 + 8.1 29.5 + 10.6 aSignificantly higher than 3hr post (P = 0.01, observed power = 0.08). bSignificant main effect across time for all conditions (P < 0.001). cSignificantly higher than post (P = 0.003) and 3 hr post (P < 0.001). dSignificantly lower than pre (P < 0.001) and post (P = 0.03).

Table 3.2. Plasma Electrolytes for Experimental Conditions (M + SD) Aggregate Placebo Npx Heat NpxHeat

Pre 134.3 + 4.6 133.5 + 5.7 133.2 + 4.5 133.5 + 3.2 135.0 + 2.9 PNa+ Post 133.5 + 3.2 133.3 + 2.9 131.5 + 4.8 132.2 + 5.6 134.8 + 3.9 (mmol/L) 3 hr post 134.3 + 3.1 133.1 + 1.9 133.5 + 0.3 132.9 + 4.7 135.3 + 2.7

Pre 3.9 + 0.4 3.9 + 0.6 4.1 + 0.4 3.9 + 0.2 3.6 + 0.1 PK+ Post 4.2 + 0.4 4.2 + 0.7 4.4 + 0.3 3.9 + 0.1 4.2 + 0.2 (mmol/L)a 3 hr post 3.9 + 0.2 3.8 + 0.3 3.8 + 0.0 4.0 + 0.3 3.9 + 0.3

aSignificant main effect across time (P = 0.02, observed power = 0.08).

3 Days – 1 Day Prior Data Collection (4 Trials) Information to Data Collection Session Post- 3hrs Post- Arrive to Lab Cycling Trial Informed Consent 3 Days Exercise Exercise Form Begin diet and Health and Injury physical activity Confirm 2 pills Continuous Blood Water ad History log taken HR Urine libitum Questionnaire No vigorous Give 3rd pill 3.5 mL/kg BM Post Graded cycling exercise Baseline water every BP and HR Blood V̇ O2max test 1 Day Blood 15 min Urine Experimental Written and Urine BM Condition verbal Take BM BP and HR 61 assignments Home HR and BP Instructions 2 pills

Figure 3.1. Experimental Procedures Schematic. Blood measures include plasma osmolality, plasma sodium concentration, and plasma potassium concentration. Urine measures include urine osmolality and urine specific gravity. Abbreviations: BM = body mass; BP = blood pressure; HR = heart rate.

CHAPTER 4

NAPROXEN EFFECTS ON GASTROINTESTINAL DISTRESS AND PERFORMANCE DURING

CYCLING IN THE HEAT1

1Dawn M. Emerson, J. Mark Davis, Stephen CL Chen, Toni M. Torres-McGehee, J. Larry Durstine, Charles C. Emerson, Craig E. Pfeifer, Joseph D. Bivona, Justin V. Stone. To be submitted to Med Sci Sport Exerc.

ABSTRACT

Purpose: Our primary objective was to determine effects of naproxen on gastrointestinal

(GI) distress symptoms and performance in hydrated humans cycling in the heat. Methods:

We utilized a double-blind, randomized and counterbalanced, cross-over design.

Participants were 11 moderately endurance trained volunteers (6 male, 5 female; age = 27.8

+ 6.5 yrs, weight = 79.1 + 17.9 kg, height = 177 + 9.5 cm, and V̇ O2max = 41.4 + 5.7 ml/kg).

Independent variables were placebo or naproxen (one 220 mg pill every 8 hrs for 24 hrs)

and hot or ambient environment. Dependent variables were max heart rate (HR), rate of

perceived exertion (RPE), distance, GI symptoms, and fecal occult blood. Measures were

taken pre-, during, and post-90 min cycling. Four trials: 1) placebo and ambient (Control);

2) placebo and heat (Heat); 3) naproxen and ambient (Npx); and 4) naproxen and heat

(NpxHeat) were completed and separated by 7 days minimum. Results: No statistically

significant differences in RPE, max HR, distance, fecal occult blood, or GI symptoms

between conditions were found. Average max HR was higher during Npx (176.2 + 15 bpm)

than Control (175.7 + 14.2 bpm) and NpxHeat (179.0 + 18.0 bmp) than Heat (177.8 + 18.2

bpm). Mean distance covered was highest during Npx (3.3 + 0.8 miles) and lowest in

NpxHeat (2.8 + 0.8 mile). Gastrointestinal symptoms occurred during exercise in 64% of

trials. Of the reported GI symptoms during ambient trials, 10% were serious compared to

7% in Heat and 1% in NpxHeat. Dizziness and headache during exercise were only

reported during placebos. Conclusion: Acute naproxen intake did not significantly affect

performance or GI distress during 90 min cycling. The potential interaction between

naproxen and heat stress, as indicated by higher HR and lower distance traveled, warrants

further research. Key Words: NSAIDs, gastrointestinal bleeding, heart rate, perceived exertion, cycling

INTRODUCTION

A high prevalence of prescription and over the counter non-steroidal anti- inflammatory drug (NSAID) use among various age groups, sport types, and competition levels exists.(38,40,41) These drugs are often used prophylactically or as treatment following injury.(11,39,40) For example, individuals may take NSAIDs in order to

“continue playing” or in attempt to mitigate pain and inflammation during activity. Because

of their association with anti-inflammatory and analgesic effects, there is a perception

NSAIDs can increase performance during physical activity.(40)

Acetaminophen has been found to enhance performance by alleviating pain,(12,23)

but research on NSAIDs is limited and results are inconclusive. Existing literature

primarily focuses on aspirin, males, and short, intense exercise bouts in ambient

environments.(6,8,15,18) For example, using aspirin (dose = 20 mg/kg of body weight) 1 hr prior to exercise did not significantly affect power output, rate of perceived exertion

(RPE), heart rate (HR), or alleviate muscle pain during a ramped maximum exertion cycling protocol.(6) Similarly, a lower aspirin dose (10 mg/kg) 1 hr prior to maximum lower extremity resistance exercises increased RPE and perceived leg pain compared to placebo.(18) Gilbert et al.(15) investigated an acute aspirin dose (975 mg 1 hr prior) and chronic (975 mg 3 times a day for 4 days) on max graded treadmill runs. Acute aspirin significantly increased RPE, while chronic significantly increased post-exercise lactate,

RPE, hematocrit, and fatigue.(15) Lastly, ibuprofen (1.2 g) 1 hr prior to treadmill running to fatigue did not improve time to exhaustion or RPE and resulted in no significant change

to blood lactate or HR.(8) From these results, NSAIDs appear to have little effect and, in

some instances, may degrade performance.

NSAIDs may also negatively affect performance through physiological mechanisms such as damage to the gastrointestinal (GI) tract,(22) cardiovascular system, and kidneys.(4) Severity for adverse NSAID effects varies depending on dosage, length of use, formula, and individual risk factors (eg, sensitivity to the drug, previous adverse

events, and health status).(22) However, in general, NSAIDs are known to cause ulcers,

perforations, and hemorrhaging along the GI tract.(14,22) Symptoms of GI distress (eg,

nausea, cramping, diarrhea, pain, and bloating) can be subjective and highly variable

between individuals. Further, GI distress can be caused by factors such as stress, fatigue,

and diet.(16,25,28) NSAIDs also increase cardiovascular strain by inducing

vasoconstriction and increasing hypertension in peripheral and renal blood vessels.(4)

Independently or concurrently, these NSAID driven events could decrease performance.

Our primary aim was to determine effects of an acute over the counter dose of naproxen on GI distress symptoms and performance measures during moderate-intense endurance exercise in a hot environment. A secondary aim was to investigate potential gender differences. We hypothesized naproxen would induce significantly more GI distress and decrease performance compared to placebo controls. We also hypothesized GI distress and performance decrements would not be different between males and females.

METHODS

Participants

As part of a larger study, 11 participants (5 male, 6 female; age = 27.8 + 6.5 yrs,

weight = 79.1 + 17.9 kg, and height = 177 + 9.5 cm) were recruited from the university and

local community. Participants read and signed an Institutional Review Board approved

informed consent form (Appendix A) prior to participation. To be included in the study

participants completed a health and injury history questionnaire (Appendix B). Participants

were free from cardiovascular, respiratory, and metabolic disorders; musculoskeletal

disorders preventing cycling exercise; fluid and electrolyte balance disorders; and GI and

swallowing disorders. Questions also asked about current medication use. Females were

asked additional questions on menstrual cycle. Participants completed a graded cycling

V̇ O2max test (Appendix E.2) to determine qualification as moderately trained (male V̇ O2max

between 35-40 mL/kg; female V̇ O2max between 32-40 mL/kg).(27)

Experimental Conditions

Participants completed all 4 experimental conditions in a randomized, counter-

balanced order with 7 days minimum between each trial. The 4 experimental conditions

were: 1) placebo and ambient (Control); 2) placebo and heat (Heat); 3) naproxen and

ambient (Npx); and 4) naproxen and heat (NpxHeat). A 24 hr dose (3 capsules) of placebo

(cellulose) or naproxen (220 mg naproxen sodium/capsule) was given to participants prior

to exercise. Participants consumed 1 capsule at 16 hrs, 8 hrs, and 0 hrs prior to data

collection. All participants were given specific take home instructions (Appendix C.4) to

follow 24 hrs prior to data collection, including directions for taking each capsule

according to the manufacturer’s directions. All experimental trials took place in an

environmental chamber (hot = 35.7 + 1.3°C, 53.2 + 3.2% relative humidity) or laboratory

(ambient = 22.7 + 1.8°C, 52.4 + 5.5% relative humidity).

Instruments and Protocols

Cycle Exercise Protocol. Participants completed a 90 min cycling protocol on a

stationary bike (Monark Ergomedic 828E, Monark Exercise AB, Vansbro, Sweden).

Participants began with a 3 min warm followed by 80 min at a steady HR equivalent to

70% V̇ O2max.(35) The final 10 min were performed at max effort and the protocol concluded with a 5 min cool down.

Rate of Perceived Exertion. The Borg Scale measured participants’ RPE pre-,

every 15 min during, and post-exercise.

Cardiovascular. To ensure participants remained at safe limits and assist with

maintaining the target HR during 80 min stead state cycling, HR was continuously

monitored and recorded every 5 min using Polar HR monitors (Polar Electro Inc., Lake

Success, NY). To determine effects on performance, we measured max HR at the end of

the 10 min max effort.

Gastrointestinal Symptom Index (Appendix D). Gastrointestinal distress was

assessed using a previously designed symptom questionnaire.(31,32) The index is divided

into 3 sections: 1) upper abdominal problems (heart burn, reflux, belching, bloating,

stomach pain/cramping, nausea, vomiting); 2) lower abdominal problems (intestinal/lower

abdominal pain/cramping, flatulence, urge to defecate, side aches/stitch, loose stool,

diarrhea); and 3) systemic problems (dizziness, headache, muscle cramps, urge to

urinate).(31) Symptoms are scored on a 10-point scale (0 = no problems at all and 9 = the

worst it has ever been). A score of > 4 is considered “serious”.(32) Questionnaires were

administered pre-, post-, and 3 hrs post-exercise. An additional questionnaire was

administered at the time of the post-exercise fecal sample (~ day post).

To determine GI symptoms during exercise, a scale was developed based on the GI

symptom questionnaire. Participants were verbally asked every 15 min “Are you currently

experiencing GI symptoms?” If yes, he/she was asked to verbalize the symptom and rank

the severity on a 10-point scale (0 = no problems at all and 9 = the worst it has ever been).

Fecal Occult Blood. To determine GI bleeding, fecal occult blood (FOB) was

measured using take home kits (Fisher HealthCareTM Sure-VueTM Fecal Occult Blood Slide

Tests System, Thermo Fisher Scientific Inc., Waltham, MA). For each experimental trial,

participants provide the first stool sample after initiating naproxen/placebo and the first

stool sample following the exercise protocol.

Hydration Measures. Participants were required to be euhydrated prior to

beginning data collection. Hydration status was determined using urine specific gravity

(Usg). Urine was obtained pre-, post-, and 3 hrs post-exercise. A handheld clinical refractometer (model REF 312, Atago Company Ltd., Tokyo, Japan) measured Usg.

Euhdyration was defined as Usg < 1.020.(34) To maintain hydration during exercise,

participants were provided 3.5 ml/kg of water to consume every 15 min.

Diet and Activity Logs. To control potential effects on GI distress and

performance, participants were asked to track diet and physical activity for 3 days prior and 1 day after data collection using an online nutrition software FoodProdigyTM (ESHA

Research, Salem, OR). Participants were asked to mimic dietary and physical activity

habits during the 24 hrs prior to each data collection session. Additionally, participants

were instructed to refrain from eating red meat during the 3 days prior and 1 day after due

to the potential to skew FOB tests.

Experimental Procedures

Information Session. After consenting participants were determined free of

disqualifying medical conditions and verified to be moderately trained, they were randomly

assigned to an experimental condition order by a research assistant. Participants and primary investigators were blind to whether participants were completing the naproxen or placebo groups. Participants were familiarized with the online diet and activity log and the

V̇ O2max test was used to familiarize individuals to the stationary cycle. Participants were instructed to not take any pain or inflammation medications during the course of the study.

Pre-Data Collection. Participants were sent an email with instructions for completing the diet and physical activity log 72 hrs before data collection. Participants were instructed to refrain from intense, vigorous exercise for 48 hrs.(29) Twenty-four hrs prior, participants were provided written instructions for taking 2 capsules (placebo or naproxen) and directions for diet, hydration, and activity. Participants were instructed to consume a small meal at least 2 hrs prior to arriving to the laboratory to ensure food passed through the stomach.

Data Collection Session. Upon arrival to the laboratory, participants were verbally asked if they took their 2 pills before taking the 3rd pill. Participants were instructed to provide a urine sample into a urine cup and void everything else into the toilet. Baseline

Usg was measured to ensure participants were euhydrated.

Participants were provided a HR monitor and the cycling protocol began with a 3 min warm up on the bike. Participants maintained their target HR during the 80 min steady state exercise and consumed water to maintain hydration. Every 15 min a research assistant asked participants their RPE and GI symptoms. The last 10 mins of the cycling protocol was completed at max effort. Each participant was given verbal encouragement by research assistants for each trial. At the conclusion of the max effort, participants were asked RPE and GI symptoms, HR was recorded, and distance traveled during the 10 mins of max

cycling was recorded. Following a 5 min cool down on the bike, participants completed a

post-GI symptom index and provided a urine sample. Participants then rested 3 hrs in a

semi-reclined/seated position in an ambient environment (23°C, 56% RH). Water was

consumed ad libitum and each participant was given a non-sucrose snack. At the

conclusion of the rest period, urine, BP, HR, and GI symptoms were measured.

Statistical Analysis

IBM SPSS Statistics (version XXII; IBM Corporation, Armonk, NY) was used for

all analyses. Descriptive statistics (mean and standard deviations) for all dependent

variables were calculated. Demographical (eg, age, height, weight) and dietary (eg, calories

and protein) differences were assessed using a one-way ANOVA. We used a one-way

ANOVA to determine differences between conditions for distance, max HR, and FOB.

Eight (time) x 4 (condition) repeated measures ANOVAs determined RPE differences for all data and between and within genders. Because RPE violated sphericity, Greenhouse-

Geisser corrections were used. Post hoc analysis was conducted for significant main effects with Bonferroni corrections. Using G*Power (version 3.1.9.2, Heinrich Heine University,

Dusseldorf, Germany),(10) post hoc power calculations with means and variances for HR

and distance indicated a statistical power > 0.9. Significance level was set at P < 0.05 for

all analyses.

Due to non-parametric data, we used Wilcoxon signed rank tests to identify

differences in GI symptoms pre-, post- and 3hr post-exercise within and between conditions. Wilcoxon signed rank were also used to determine differences within gender.

To reduce multiplicity, questions were sectioned into upper, lower, and systemic symptoms. Responses were averaged and analyzed. Because of the number of tests and

chances for inflation, P values are considered “interesting” rather than significant.(31,32)

We determined frequency of symptoms scored > 4 (considered “serious”) within each

condition. Kruskal-Wallis analysis determined differences between genders. To control for

other factors that may influence GI symptoms, Spearman’s rho correlations were run for

max core temperature (data presented in Chapter 2), fluid volume consumed during

exercise (data presented in Chapter 3), max HR, max RPE, and distance.

RESULTS

Table 4.1 contains participant demographics. There was no significant differences

between age, weight, and V̇ O2max for gender. Participants began experimental trials euhydrated (mean Usg = 1.012 + 0.005) and maintained euhydration throughout exercise

(1.011 + 0.008) and recovery (1.007 + 0.006). Diet log results indicated no significant

differences between conditions for calorie, fat, protein, carbohydrate, or sodium intake.

Also, no significant differences in exercise (calories burned) 24 hrs prior to data collection

existed, with all participants maintaining sedentary to low activity. No significant

differences existed within genders between experimental conditions. We found, overall,

males consumed significantly more calories, protein, and sodium than females (Appendix

F, Table F.2). Subsequent analysis identified the only significant difference between

genders within conditions occurred with protein for NpxHeat (male = 65.4 + 2.7 g and

female = 44.7 + 3.2 g, P = 0.004).

Performance

Table 4.2 shows max HR, RPE, and distance traveled during 10 min max effort for

each condition. There was an overall significant main effect (P < 0.001) for RPE over time, but no significant differences between conditions. Within gender, RPE did not significantly differ between conditions (Appendix F, Table F.3). The only difference between gender 71

and conditions occurred in NpxHeat with pre-exercise males significantly lower (7 + 1) than females (9 + 2, P = 0.03). Significant differences occurred between overall males and female RPEs. Females were significantly higher at 0 and 15 min cycling (10 + 2 vs. 8 + 2 and 11 + 2 vs. 9 + 3, respectively, P < 0.03). On the other hand, males were significantly higher at 90 min (19 + 2 vs. 18 + 2, P < 0.03).

Distance was not significantly different between conditions. On average, distance traveled was lower during heat trials compared to ambient (Table 4.2). Interestingly, mean distance was slightly higher for Npx compared to Control. This effect was not found in the hot environment, with NpxHeat having the lowest distance covered. There were no statistically significant difference between males and females for distance within or between conditions (Appendix F, Table F.4). Overall, we found males trended toward greater distance than females (3.2 + 0.7 miles vs 2.8 + 0.9 miles, P = 0.085). Similar to overall condition results, both males and females in ambient conditions averaged greater distance compared to heat. Furthermore, the Npx condition resulted in the highest average distance for both genders (Appendix F, Table F.4).

As expected, mean max HR was higher during heat trials compared to ambient, but was not significantly different (Table 4.2). Heart rate was highest during NpxHeat and lowest in the Control trial. There were no statistically significant difference within or between genders (Appendix F, Table F.4). Females’ mean max HR was highest in NpxHeat

(180.2 + 17.2 bpm) and was higher than males for both heat trials. Male max HR was highest during the Npx condition (179.5 + 15.1 bpm).

GI Symptoms and FOB

Mean and max scores for each upper, lower, and systemic GI symptom were totaled for each time point (pre- to day-post) and are presented in Table 4.3. Both Heat and

NpxHeat had higher averages and max scores compared to ambient conditions. Urge to

urinate was the most frequent symptom reported. Reflux/heartburn, vomiting, abdominal

pain, and diarrhea were only reported during heat trials.

Appendix F, Table F.5 shows aggregate upper, lower, and systemic GI scores for

each time point with Wilcoxon results. Heat resulted in higher mean upper GI symptom

scores post-exercise (0.3 + 0.3). Higher mean and serious systemic post-exercise scores

occurred in Control (0.9 + 0.9, 6% serious), Npx (0.6 + 0.6, 6% serious), and Heat (0.7 +

0.9, 3% serious) compared to NpxHeat (0.3 + 0.4, 0% serious). Control continued to

experience more frequent and serious lower and systemic symptoms at 3 hrs post-exercise and was greater than any other condition. Day-post results showed Heat experienced higher mean and serious scores for upper (1.3 + 2.5, 18% serious), lower (1.9 + 12.4, 6% serious)

and systemic (1.3 + 2.2, 5% serious). Only NpxHeat resulted in more serious lower GI

symptoms day post (12%).

There were no significant differences for GI symptoms between conditions within

genders. The only significant differences between genders occurred at 3 hrs within the Heat

condition, χ2(1) = 4.4, P = 0.036, with a mean rank 4.5 for males and 7.8 for females. There were no positive FOB tests pre-exercise. One positive FOB test indicated GI bleeding post-

exercise in the Npx condition. However, this test was likely a false positive, since it

occurred in a menstruating participant.

During Exercise

Overall, GI symptoms occurred during exercise in 64% of trials. Only 1 participant experienced no GI symptoms during any trial. Eight-two percent of participants reported at least 1 symptom during Control, 73% during Npx, 45% during Heat, and 55% during

NpxHeat. Mean and max GI symptom scores during exercise are presented in Table 4.4.

Urge to urinate and nausea was most frequent for all conditions and highest during ambient trials. Greatest percentage of serious scores (10%) occurred for systemic symptoms, specifically urge to urinate, during Control and Npx (Appendix F, Table F.6). NpxHeat had the least systemic symptoms reported (0.0 + 0.8, 1% serious). Dizziness and headaches were reported only during placebo conditions. Though lower GI symptoms were frequently reported during exercise for all conditions, none were classified as serious. Upper GI symptoms were commonly reported with Heat (0.6 + 1.0, 3% serious), NpxHeat (0.3 + 0.9 and 2%), Control (0.3 + 0.6, 1%), and Npx (0.4 + 0.7, 1%). Nausea and vomiting were the only reported serious upper GI symptoms. There were no significant differences for GI symptoms during exercise either within or between genders.

Correlations

We identified no significant correlations for GI symptoms to fluid volume, core temperature, max RPE, or max HR. We found a significant positive correlation between max RPE and HR for Control (ρ = 0.8, P = 0.002), Npx (ρ = 0.8, P = 0.006), and Heat (ρ

= 0.6, P = 0.43), but not for NpxHeat. A significant positive correlation was found for max core temperature and HR only during Control (ρ = 0.7, P = 0.025). Distance traveled was significantly correlated to max RPE during Npx (ρ = 0.6, P = 0.044) and to core temperature during NpxHeat (ρ = 0.7, P = 0.028). During Heat, we found a significant

positive correlation between lower and upper GI symptoms (ρ = 0.9, P = 0.001) and lower and systemic GI symptoms (ρ = 0.7, P = 0.03).

We identified several significant correlations within genders. Distance and max HR in females were strongly correlated during the Control (ρ = 1.0, P < 0.001), but this did not occur in the males. Likewise, distance was strongly correlated to max HR (ρ = 1.0, P <

0.001) and RPE (ρ = 1.0, P < 0.001) during Npx, but this was not seen in males. During

Npx, we found male RPE was negatively correlated with upper GI symptoms (ρ = -1.0, P

< 0.001). In the Heat, female, but not male, RPE was strongly correlated to distance (ρ =

1.0, P < 0.001) and fluid volume (ρ = 1.0, P = 0.005). Finally, within NpxHeat, female distance was significantly correlated to upper GI symptoms (ρ = 0.9, P = 0.041) and HR (ρ

= 1.0, P = 0.005). On the other hand, in males, core temperature was strong correlated to distance (ρ = 0.8, P = 0.036) and systemic GI symptoms (ρ = 0.9, P = 0.029).

DISCUSSION

Extensive evidence shows NSAIDs negatively affect the GI and cardiovascular systems,(13,17,20,22) yet there remains a high prevalence of NSAIDs use before and during exercise.(11,38-40) Often, NSAIDs are taken to prevent or alleviate pain and inflammation and, seemingly, these effects would increase performance. Ours results show a 24 hr dose of naproxen does not improve performance, as measured by RPE, distance, and max HR. We also found compared to placebos, naproxen did not significantly increase

GI symptoms pre-, during, or post-exercise and these responses were not different between males and females.

Performance

Like previous studies using aspirin(6,15,18) and ibuprofen,(8) naproxen did not

improve performance. However, contrary to our hypothesis, we did not find naproxen

significantly decreased performance variables. A potential explanation for the lack of

statistical significance is using a low dose. Variation in dosage is partly due to differences

in cyclooxygenase (COX) selectivity and half-life. Compared to aspirin and ibuprofen, naproxen is more selective toward inhibiting COX 2, which is produced by tissues locally in response to damage(21) and is a key mediator of pain and inflammation.(1) Through higher selectivity, naproxen is considered a more effective anti-inflammatory and analgesic than other NSAIDs.(2) Naproxen also has a longer half-life, 10-20 hrs, meaning individuals can take less doses than aspirin (2-3 hr half-life) and ibuprofen (2-4 hr half-life).(5) With these considerations, higher aspirin and ibuprofen doses are recommended to elicit anti- inflammatory and analgesic effects, and therefore higher doses are seen in literature.(6,8,15,18) Increasing the naproxen dose (ie, prescription strength) and extending time used (ie, more than 24 hrs) could elicit different, more significant responses. This thought is supported when examining acute versus chronic aspirin use, with acute eliciting significantly increased RPE, while chronic significantly affected RPE, lactate, hematocrit, and fatigue.(15)

Our participants were euhydrated throughout exercise; therefore, increased HR was not dehydration induced, but rather associated with experimental conditions. When examining heat trials, NpxHeat averaged less miles with a higher HR and RPE compared to Heat. This same increase in cardiovascular strain was seen when we examined gender differences. Males averaged the same mileage in heat trials, but they experienced higher

max HR with naproxen. Females covered the least amount of distance and experienced their highest HR during NpxHeat. Keeping in mind NSAID effects on the cardiovascular system (vasoconstriction and increased BP) and the cardiovascular systems’ response to intense exercise under thermal stress (increased HR to maintain cardiac output),(24) our results suggest combining naproxen and heat could increase cardiovascular strain and max

RPE, resulting in less distance.

Slightly different results were found among the ambient conditions. Naproxen resulted in the greatest distance covered with no change in RPE and a slightly higher HR.

Both males and females averaged their greatest distance during the Npx trial compared to

Control. Males experienced their highest max HR and RPE during Npx, suggesting they were cycling at a higher workload than during Control. While females averaged more mileage during Npx, their max HR was lower than any other trial. The slight differences between heat and ambient suggests environment may impact the effects of naproxen on performance. To our knowledge, examining NSAIDs on performance in the heat has not been conducted. Da Silva et al.(8) used ambient conditions (18-21°C) and since other laboratory studies do not provide environment information,(6,15,18) we assume these were also conducted in ambient conditions.

Our high correlation between max HR and max RPE in all conditions, except for

NpxHeat, suggests our participants were able to accurately assess their RPE and were exhibiting max effort at 90 min. During minutes 30 to 80 of steady state, RPE did not differ between genders. This is consistent with genders working at a relative % V̇ O2max during treadmill running and cycling,(33) but contrasts Cook et al,(7) who found females reported lower RPE than males during cycling at given percentages of peak power output. Once

cycling reached peak power output, males reported significantly higher RPE than females

(19.6 + 0.9 and 18.7 + 1.4, respectively, P < 0.008).(7) The significantly higher aggregate

values for our male participants compared to females reflects at max effort males report

higher RPE, but when working at a relative percentage of V̇ O2max males and females

respond similarly. Moreover, this was not influenced by taking naproxen or by the

environment.

Gastrointestinal Distress

Intense physical exercise and heat stress can induce GI symptoms and is heavily

influenced by factors such as previous and current medical history, medication, nutrition,

and exercise mode. Typically, endurance exercise elicits more GI symptoms than

anaerobic, and running generally induces greater symptoms than cycling.(29,30) The

etiology for GI distress is not well understood, but is suggested to be attributed to

mechanical vibrations,(9) GI ischemia-reperfusion,(3,37) and inflammatory

responses.(3,19) Prevalence for GI symptoms during exercise in our study is similar to

ultra-distance runners (60%).(36) Type of symptoms (ie, nausea, diarrhea, and vomiting)

was also similar to runners.(36) However, considering the prevalence of GI bleeding during

running,(25) we were surprised to find a lack of positive FOB, particularly with the

NpxHeat. We likely attenuated the incidence for GI bleeding by using a less impactful

exercise (cycling) and a shorter, moderate-intense exercise bout.

Contrary to our hypothesis, naproxen did not significantly increase GI distress

compared to placebo. One of the most notable findings is naproxen preventing dizziness

and headache during exercise, but not before or after exercise, in both heat and ambient trials. Compared to Control, Npx experienced more upper GI symptoms during exercise

but less systemic symptoms. Both Heat and NpxHeat experienced upper GI symptoms, but

NpxHeat, once again, experienced less systemic. The high percentage of serious scores in the ambient conditions compared to heat are probably due to the exercise intensity during

80 min cycling prior to max effort. Because participants cycled at a steady HR, during the heat trials HR naturally increased in response to thermal strain. This forced participants to cycle at a lower intensity to maintain target HR. Less intensity explains the lower percentage of serious scores and symptoms in the heat. Additionally, urge to urinate during heat trails was likely mitigated by increased fluid needs in the hot environment (ie, sweating). Interestingly, higher frequency and serious scores were reported day-post during

Heat and NpxHeat trials compared to ambient. These data suggest lingering effects from exercise in a hot environment, which was, to some extent, alleviated by naproxen. We did not assess physiological measures day-post, but exercise and thermal stress induce inflammatory responses(35) that can last hours to days after.(26) Increased inflammation could explain higher GI symptoms day-post and is an important consideration for individuals exercising on consecutive days.

Despite slight differences in cardiovascular and performance measures between genders, we found no differences in GI symptoms. Furthermore, GI symptoms in females were not different during menstruation. In our study, GI symptoms were not correlated with the amount of fluid ingested, core temperature, max HR, or max RPE. Instead, GI symptoms were most likely induced by exercise, environment, and/or naproxen.

Performance variables seemed to be influenced more by each other, particularly between

RPE and HR. Correlations among genders were slightly different than overall. The only significant correlations among males occurred during Npx and NpxHeat. During Npx,

more GI symptoms resulted in lower RPE in males, presumably limiting their ability or desire to cycle at max effort. In NpxHeat, higher core temperatures were associated with longer mileage and more systemic GI symptoms. In females, longer distance during

NpxHeat was related to higher GI symptoms and HR. These two findings suggest the GI symptoms during NpxHeat occurred as a result of working at max rather than GI symptoms inhibiting effort, as seen with the Npx in males.

Limitations and Future Research

As with any subjective measure, we assume participants were honest when answering the GI symptom indexes. Similarly, we did not measure V̇ O2max or power output

during exercise, and therefore, must base our assumption that participants gave the same,

max effort during each trial on HR and RPE. Frequency and severity for urge to urinate

during and post-exercise was likely skewed by forcing participants to drink to maintain

hydration, making it difficult to determine actual naproxen effects. This would be mitigated

if participants drank the same fluid volume during each trial; however, participants were

allowed to consume more fluid than provided. As previously mentioned, using a constant

HR rather than V̇ O2max resulted in a lower exercise intensity during heat trials. Our results are applicable for individuals who exercise at a given HR (eg, as a way to maintain pace), but this methodology mitigated finding significance during experimental conditions.

Another potential limitation was using guaiac-based FOB to determine GI bleeding. By measuring heme-, FOB has high specificity for detecting blood from any portion of the GI tract (ie, stomach, small intestine, and colon). However, its sensitivity varies depending on brand. It is possible, due to low sensitivity, the FOB was not able to detect blood in our test samples, resulting in false negatives. An alternative testing option is using fecal

immunological test, which is not affected by diet and is highly sensitive to blood in stool.

However, this test is less specific, only being able to detect bleeding from the colon.

There are several areas for future research to examine NSAID effects on

performance and GI distress during exercise. Research should examine different dosages

(eg, prescription strength) and length of use (eg, 3 days, 7 days, 14 days). It is pertinent to

examine hypohydrated individuals, which would increase cardiovascular strain, GI

distress, and perceived exertion in both ambient and hot conditions. Using a carbohydrate

electrolyte beverage for hydration would introduce potential GI distress. Research is

needed on different exercise modes, intensity, and length. Lastly, further research is warranted in both males and females to identify potential gender differences.

Conclusion

Among hydrated, moderate-endurance trained males and females, a 24 hr over the counter dose of naproxen did not improve performance during a 90 min cycling bout in either ambient or hot conditions. Additionally, participants reported upper, lower, and systemic GI symptoms during exercise, but naproxen did not increase occurrence or severity. The trend for higher HR with lower distance, as well as correlations among males and females for GI symptoms and distance in NpxHeat, suggest combining naproxen and thermal stress could be detrimental to performance. Considering prevalent NSAID use

among physically active persons and known effects on the cardiovascular and GI systems,

further research on health and performance measures is warranted, particularly in hot environments.

REFERENCES

1. Brock TG, McNish RW, Peters-Golden M. Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2. J Biol Chem. 1999;274(17):11660-6.

2. Capone ML, Tacconelli S, Sciulli MG, et al. Human pharmacology of naproxen sodium. J Pharmacol Exp Ther. 2007;322(2):453-60.

3. Carden DL, Granger DN. Pathophysiology of ischemia-reperfusion injury. J Pathol. 2000;190:255-66.

4. Cheng HF, Harris RC. Renal effects of non-steroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors. Curr Pharm Des. 2005;11(14):1795-804.

5. Ciccone CD. Davis's Drug Guide for Rehabilitation Specialists. Philadelphia, PA: F. A. Davis; 2013.

6. Cook DB, O'Connor PJ, Eubanks SA, Smith JC, Lee M. Naturally occurring muscle pain during exercise: assessment and experimental evidence. Med Sci Sport Exerc. 1997;29(8):999-1012.

7. Cook DB, O'Connor PJ, Oliver SE, Lee Y. Sex differences in naturally occuring leg muscle pain and exertion during maximal cycle ergometry. Intern J Neuroscience. 1998;95:183-202.

8. Da Silva E, Pinto RS, Cadore EL, Kruel LF. Nonsteroidal anti-inflammatory drug use and endurance during running in male long-distance runners. J Athl Train. 2015;50(3):295-302.

9. de Oliveira EP, Burini RC. The impact of physical exercise on the gastrointestinal tract. Curr Opin Clin Nutr Metab Care. 2009;12(5):533-8.

10. Faul F, Erdfelder E, Lang A-G, Buchner A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods. 2007;39(2):175-91.

11. Feucht CL, Patel DR. Analgesics and anti-inflammatory medications in sports - use and abuse. Pediatr Clin N Am. 2010;57:751-74.

12. Foster J, Taylor L, Chrismas BC, Watkins SL, Mauger AR. The influence of acetaminophen on repeated sprint cycling performance. Eur J Appl Physiol. 2014;114(1):41-8.

13. Garcia Rodriguez LA, Gonzalez-Perez A. Long-term use of non-steroidal anti- inflammatory drugs and the risk of myocardial infarction in the general population. BMC Med. 2005;3:17-22.

14. Gibson GR, Whitacre EB, Ricotti CA. Colitis induced by nonsteroidal anti- inflammatory drugs. Arch Intern Med. 1992;152:625-32.

15. Gilbert JA. Acute and chronic effect of apirin on selected endurance variables. Sports Med Train Rehab. 1995;6(4):299-307.

16. Goodman CC, Fuller KS. Pathology: Implications for the Physical Therapist. 3rd ed. St. Louis, MO: Saunders Elsevier; 2009.

17. Haag MDM, Bos MJ, Hofman A, Koudstaal PJ, Breteler MMB, Stricker BHC. Cyclooxygenase selectivity of nonsteroidal anti-inflammatory drugs and risk of stroke. Arch Intern Med. 2008;168(11):1219-24.

18. Hudson GM, Green JM, Bishop PA, Richardson MT. Effects of caffeine and aspirin on light resistance training performance, perceived exertion, and pain perception. J Strength Cond Res. 2008;22(6):1950-7.

19. Jeukendrup AE, Vet-Joop K, Sturk A, et al. Relationship between gastro-intestinal complaints and endotoxaemia, cytokine release and the acute-phase reaction during and after a long-distance triathlon in highly trained men. Clin Sci. 2000;98:47-55.

20. Lewis SC, Langman MJS, Laporte J-R, Matthews JNS, Rawlins MD, Wiholm B-E. Dose–response relationships between individual nonaspirin nonsteroidal anti- inflammatory drugs (NANSAIDs) and serious upper gastrointestinal bleeding: a meta-analysis based on individual patient data. Br J Clin Pharmacol. 2002;54:320-6.

21. Li H, Edin ML, Gruzdev A, et al. Regulation of T helper cell subsets by cyclooxygenases and their metabolites. Prostaglandins Other Lipid Mediat. 2013;104-105:74-83.

22. Masso Gonzalez EL, Patrignani P, Tacconelli S, Garcia Rodriguez LA. Variability among nonsteroidal antiinflammatory drugs in risk of upper gastrointestinal bleeding. Arthritis Rheum. 2010;62(6):1592-601.

23. Mauger AR, Taylor L, Harding C, Wright B, Foster J, Castle PC. Acute acetaminophen (paracetamol) ingestion improves time to exhaustion during exercise in the heat. Exp Physiol. 2014;99(1):164-71.

24. McArdle WD, Katch FI, Katch VL. Exercise Physiology: Energy, Nutrition, and Human Performance. Fifth ed. Baltimore, MD: Lippincott Williams & Wilkins; 2001.

25. Moses FM. The effect of exercise on the gastrointestinal tract. Sports Med. 1990;9(3):159-72.

26. Pedersen BK, Rohde T, Ostrowski K. Recovery of the immune system after exercise. Acta Physiol Scand. 1998;162:325-32.

27. Pescatello LS, ed ACSM's Guidelines for Exercise Testing and Prescription. 9th ed. Baltimore, MD: Lippincott Williams & Wilkins; 2014. Arena R, ed. Exercise Testing.

28. Peters HPF, Bos M, Seebregts L, et al. Gastrointestinal symptoms in long-distance runners, cyclists, and triathletes: prevalence, medication, and etiology. Am J Gastroenterol. 1999;94(6):1570-81.

29. Peters HPF, Wiersma WC, Akkermans LM, et al. Gastrointestinal mucosal integrity after prolonged exercise with fluid supplementation. Med Sci Sport Exerc. 2000;32(1):134-42.

30. Peters HPF, Wiersma WC, Koerselman J, et al. The effect of a sports drink on gastroesophageal reflux during a run-bike-run test. Int J Sports Med. 1999;20:65-70.

31. Pfeiffer B, Cotterill A, Grathwohl D, Stellingwerff T, Jeukendrup AE. The effect of carbohydrate gels on gastrointestinal tolerance during a 16-km run. Int J Sport Nutr Exerc Metab. 2009;19:485-503.

32. Pfeiffer B, Stellingwerff T, Hodgson AB, et al. Nutritional intake and gastrointestinal problems during competitive endurance events. Med Sci Sport Exerc. 2012;44(2):344-51.

33. Robertson RJ, Moyna NM, Sward KL, Millich NB, Goss FL, Thompson PD. Gender comparison of RPE at absolute and relative physiological criteria. Med Sci Sport Exerc. 2000;32(12):2120-9.

34. Sawka MN, Burke LM, Eichner ER, Maughan RJ, Montain SJ, Stachenfeld NS. American College of Sports Medicine position stand. Exercise and fluid replacement. Med Sci Sport Exerc. 2007;39(2):377-90.

35. Starkie RL, Hargreaves M, Rolland J, Febbraio MA. Heat stress, cytokines, and the immune response to exercise. Brain Behav Immun. 2005;19(5):404-12.

36. Stuempfle KJ, Hoffman MD, Hew-Butler T. Association of gastrointestinal distress in ultramarathoners with race diet. Int J Sport Nutr Exerc Metab. 2013;23:103-9.

37. ter Steege RW, Kolkman JJ. Review article: the pathophysiology and management of gastrointestinal symptoms during physical exercise, and the role of splanchnic blood flow. Aliment Pharmacol Ther. 2012;35(5):516-28.

38. Tscholl P, Alonso JM, Dolle G, Junge A, Dvorak J. The use of drugs and nutritional supplements in top-level track and field athletes. Am J Sports Med. 2010;38(1):133- 40.

39. Warden SJ. Prophylactic use of NSAIDs by athletes: a risk/benefit assessment. Phys Sportsmed. 2010;1(38):1-7.

40. Warner DC, Schnepf G, Barrett MS, Dian D, Swigonski NL. Prevalence, attitudes, and behaviors related to the use of nonsteroidal anti-inflammatory drugs (NSAIDs) in student athletes. J Adolescent Health. 2002;30:150-3.

41. Wharam PC, Speedy DB, Noakes TD, Thompson JM, Reid SA, Holtzhausen LM. NSAID use increases the risk of developing hyponatremia during an Ironman triathlon. Med Sci Sport Exerc. 2006;38(4):618-22.

Table 4.1. Participant Demographics (M + SD) Aggregate Male Female (N = 11) (N = 6) (N = 5) Age (yrs) 27.8 + 5.7 28.7 + 5.3 26.8 + 6.5 Weight (kg) 79.1 + 17.9 88.4 + 14.0 67.9 + 16.5 Height (cm) 177.0 + 9.5 183.2 + 5.3a 169.5 + 7.8 Body fat (%) 15.2 + 8.3 10.7 + 4.0a 20.6 + 9.1

V̇ O2max 41.4 + 5.7 43.6 + 5.3 38.7 + 5.3 (mL/kg) aSignificant difference between genders (P < 0.04).

Table 4.2. Performance Measures during Cycling for Experimental Condition (M + SD)

Control Npx Heat NpxHeat Distance (miles) 3.2 + 0.8 3.3 + 0.8 2.9 + 0.8 2.8 + 0.8 Max HR (bpm) 175.7 + 14.2 176.2 + 15.0 177.8 + 18.2 179.0 + 18.0 RPE Pre 8 + 2 9 + 3 9 + 3 8 + 2 80 min 13 + 2 13 + 3 12 + 2 12 + 2 Post 19 + 2 19 + 2 18 + 2 19 + 2

No significant differences between trials.

Table 4.3. Mean and Maximum Gastrointestinal Symptom Scores for Experimental Conditions

Control Npx Heat NpxHeat Symptoms Max M + SD Max M + SD Max M + SD Max M + SD Upper Reflux/Heartburn - - - - 6 0.1 + 0.9 - - Belching 1 0.1 + 0.4 - - 6 0.2 + 0.9 4 0.1 + 0.6 Bloating 5 04 + 1.2 2 0.2 + 0.4 6 0.5 + 1.3 2 0.2 + 0.8 Stomach pain 2 0.3 + 0.6 2 0.2 + 0.5 6 0.4 + 1.3 2 0.3 + 1.1 Vomiting - - - - 2 0.2 + 1.1 2 0.2 + 1.1 Nausea 3 0.2 + 0.5 1 0.0 + 0.2 6 0.5 + 1.4 3 0.3 + 1.2 Lower 88

Intestinal cramps 2 0.3 + 0.7 1 0.0 + 0.2 8 0.5 + 1.6 2 0.2 + 1.2 Flatulence 3 0.3 + 0.8 2 0.1 + 0.5 4 0.3 + 0.9 5 0.3 + 1.0 Urge to defecate 5 0.4 + 1.0 4 0.2 + 0.7 6 0.5 + 1.5 7 0.3 + 1.4 Abdominal pain - - - - 2 0.1 + 0.5 - - Loose stool/Diarrhea - - - - 4 0.4 + 1.5 7 0.4 + 1.7 Systemic

Dizziness 3 0.1 + 0.5 2 0.1 + 0.3 3 0.2 + 0.7 3 0.2 + 0.6 Headache 5 0.6 + 1.0 2 0.1 + 0.4 5 0.3 + 0.9 3 0.2 + 0.6 Muscle cramps 3 0.1 + 0.4 1 0.0 + 0.2 6 0.1 + 0.9 - - Urge to urinate 8 0.8 + 1.9 7 0.9 + 1.7 8 0.9 + 1.7 5 0.5 + 1.2

Based on aggregate scores for all symptoms reported at pre-, post-, 3 hrs post-, and day post-exercise.

Table 4.4. Mean and Maximum Gastrointestinal Symptom Scores for Experimental Conditions during Exercise

Control Npx Heat NpxHeat Symptoms Max M + SD Max M + SD Max M + SD Max M + SD Upper Reflux/Heartburn - - 2 0.0 + 0.2 - - - - Belching ------Bloating - - 3 0.1 + 0.3 4 0.1 + 0.5 2 0.0 + 0.2 Stomach pain - - 2 0.1 + 0.4 4 0.1 + 0.6 - - Vomiting - - - - 5 0.1 + 0.5 7 0.1 + 0.7 Nausea 8 0.2 + 0.9 8 0.1 + 0.8 7 0.1 + 0.9 7 0.1 + 0.7 Lower 89

Intestinal cramps 3 0.3 + 0.9 - - 4 0.1 + 0.6 3 0.1 + 0.4 Flatulence 1 0.0 + 0.1 ------Urge to defecate - - 4 0.1 + 0.4 4 0.0 + 0.4 4 0.1 + 0.6 Abdominal pain ------Loose stool/Diarrhea ------Systemic Dizziness 4 0.1 + 0.5 - - 5 0.1 + 0.5 - - Headache 4 0.2 + 0.9 - - 4 0.0 + 0.4 - - Muscle cramps ------Urge to urinate 8 0.8 + 1.9 8 0.9 + 1.9 7 0.4 + 1.5 5 0.1 + 0.7

CHAPTER 5

PROPOSAL

INTRODUCTION

During intense exercise, especially in hot, humid environments, a number of body

systems must work concurrently to cool the core, maintain muscle function, and maintain

vital cardiovascular function. The cardiovascular system’s response to exercise and thermal

stress depends on the intensity of each, but in general responds by increasing peripheral

blood flow and shunting blood away from the gut toward working muscles. Peripheral

vasodilation allows the release of metabolic heat produced during exercise, and shunting

blood to working muscles allows the individual to maintain the given activity. As exercise

and thermal load increase, the system must prioritize whether to maintain blood pressure

(BP), skin blood flow, or muscle function.1 The cardiovascular system can temporarily

maintain BP, and overall cardiac output, by increasing heart rate (HR). However,

maximum exertion and thermal overload, especially if combined with dehydration, can

exceed this ability.1 Upon cardiovascular compromise, and with the gastrointestinal (GI)

tract in a state of ischemia from decreased blood flow, core temperature (Tc) will increase

and systemic inflammatory response (SIR) occurs.2,3

Prevalence of previous illness and GI distress during exercise and thermal stress

suggests the GI tract and immune system impact the development of exertional heat illness

(EHI), especially exertional heat stroke (EHS). Symptoms of EHS (vomiting, dehydration,

multi-system organ failure, etc.)4 are similar to SIR5 and may be driven by GI distress.

Gastrointestinal barrier dysfunction leads to the entry of bacterial lipopolysaccharide (LPS) into the circulation (known as endotoxemia), release of inflammatory cytokines, and a

number of other factors that compromise the immune system. Consequently, SIR and

thermal stress intolerance occurs, which dramatically increases the risk for EHS and death.

STATEMENT OF THE PROBLEM

Extensive scientific literature exits examining potential risk factors,6,7 ways to

prevent,8,9 and ways to treat EHS.10,11 Unfortunately, preventable deaths from EHS still

occur.12 One potential contributing factor to EHS development may be non-steroidal anti-

inflammatory drugs (NSAIDs). Research has shown NSAIDs increase GI permeability

during exercise in thermoneutral environments,13-15 cause GI distress16,17 and induce

inflammatory responses.18 Despite knowing GI distress and inflammation are associated

with EHS and NSAIDs, limited research has evaluated NSAIDs’ effects on Tc during

exercise under thermal stress.

SPECIFIC AIMS AND HYPOTHESIS

Overall Hypothesis. Naproxen (NSAID) will increase GI permeability and inflammation

during exercise in the heat, and will be associated with increased GI distress and

thermoregulatory strain (Figure 5.1). This overall hypothesis is tested under three specific

aims.

Specific Aim 1. Determine the effects of naproxen on the GI tract and inflammation during exercise in the heat.

Hypothesis 1.1. GI tract permeability is greater in participants taking naproxen

compared to placebo controls during exercise in the heat.

Hypothesis 1.2. GI distress, indicated by GI symptoms (eg, nausea, cramping) and

occult fecal blood, is greater in participants taking naproxen compared to placebo

controls during exercise in the heat.

Hypothesis 1.3. Plasma concentration of inflammatory cytokines (TNF-α and IL-

6) is increased in participants taking naproxen compared to placebo controls during

exercise in the heat.

Specific Aim 2. Determine effects of naproxen on thermoregulation and performance

during exercise in heat.

Hypothesis 2.1. Participants taking naproxen will experience a faster rise in Tc

during exercise in the heat compared to placebo controls.

Hypothesis 2.2. Cardiovascular strain (HR, BP, fluid imbalance) is greater in

participants taking naproxen compared to placebo controls during exercise in the

heat.

Hypothesis 3.2. Distance covered during exercise in the heat is lower in

participants taking naproxen compared to placebo controls.

Specific Aim 3. Determine the relationship between naproxen and the GI tract and

inflammation on thermoregulation during exercise in the heat.

Hypothesis 3.1. Participants taking naproxen will have an increased Tc due to

increased GI permeability and inflammatory cytokines.

LITERATURE REVIEW

GASTROINTESTINAL TRACT

Anatomy & Physiology of Gastrointestinal Tract

The GI tract is a long, continuous, muscular tube made up of the mouth, pharynx,

esophagus, stomach, small intestine, and large intestine. Three parts make up the small

intestine: duodenum, jejunum, and ileum. The large intestine is made up of 8 parts:

transverse colon, descending colon, ascending colon, cecum, sigmoid colon, rectum,

appendix, and anal canal. The GI tract is lined by an epithelial barrier made up of 4 layers,

from deep to superficial: mucosa, submucosa, muscularis externa, and serosa.19

Stomach

The stomach is a storage area and site of chemical breakdown of food products.

When the stomach is empty, mucosa and submucosa layers fold inward to form rugae,19

allowing the stomach to expand as food is ingested. The stomach is divided into four major

regions - cardia, fundus, body, and pylorus. Closest to the heart, the cardia region contains

the gastroesophageal sphincter, which controls food entering the stomach or prevents

stomach contents from entering the esophagus.19 The fundus is the dome shaped region,

extending slightly superior and lateral to the cardia, resting against the diaphragm. The

largest region, the body, curves into the pylorus. At the distal portion of the pylorus is the

pyloric sphincter, which controls stomach contents entering the small intestine.19 Although

made up of the same epithelial and muscular layers, the 4 stomach regions vary slightly in

their function during digestion.20 As food moves in to the cardia region, there is only rippling of tissue layers. However, the muscularis layer of the body and plyorus regions are much thicker and therefore more powerful. Subsequently, food products in these regions are aggressively mixed with the gastric juice to produce chyme.19,20 Converting

solid food products to chyme, a creamy paste,19 allows for absorption of nutrients and

elimination of byproducts through the GI tract.

Gastric emptying, the time from food entering stomach to entering small intestine,

typically takes 4 hours.19 However, this time is heavily influenced by type of food, amount

of fluid, and individual health factors. Gastric emptying is faster with greater amounts of

fluid or carbohydrates. On the other hand, gastric emptying time slows down with more

solid or fatty meals.19

The stomach contains a number of defense features against bacteria and other

pathogens. A large collection of lymph nodes are responsible for fighting pathogens within

the peritoneal cavity and intraperitoneal organs.19 Gastric acid is the stomach’s key

defense. With a pH around 2, gastric acid is designed to control the amount of bacteria

present in the stomach and digest proteins.19

Small Intestine

Three unique structure modifications of small intestine epithelium allows for increased absorption. Together, the mucosa and submucosa layers are permanently folded into circular folds, or plicae circulares. These folds slow the movement of digestive product to allow for full absorption. On the outside of the mucosa cells are villi, fingerlike projections made of epithelial cells. Villi absorb nutrients into the blood stream through their dense capillary bed. Microvilli are located on the plasma membrane of mucosa cells and contain enzymes that complete digestion of carbohydrates and proteins.19

Like the stomach, the small intestine contains a number of protective factors.

Between villi are crypts of Lieberkühn. Within crypts are Paneth cells that release

lysozyme, an antibacterial enzyme that protects the small intestine from bacteria.19 Another

protection against bacteria comes from Peyer’s patches located in submucosa and

immunocytes.19,21 The small intestine contains intestinal juice. However, unlike gastric

juice, intestinal juice has a relatively neutral pH, is primarily made up of water and mucus,

and does not contain many digestive enzymes. The primary function of intestinal juice is

to protect the epithelial lining from pathogens.19

Large Intestine

Notable differences occur in the large intestine layers. Because most nutrient

absorption takes place in the small intestine, the simple columnar epithelium contains no

folds, villi, or digestive enzyme secretions. Large intestine characteristics include a thick

mucosa layer, large number of goblet cells, and a large number of deep crypts. Mucosa is

especially important to protect the wall against passing feces and acids/gases from

bacteria.19

Most bacteria that enter the large intestine have been killed by highly acidic gastric

juice or small intestine lysozymes. However, a number of bacteria enter the large intestine

and colonize; this is known as the bacterial flora. Bacteria are naturally occurring, playing

several imperative roles in maintaining human health. For instance, the bacteria flora

assists in mucosal homeostasis, repair, and glucose transporter induction; induced capillary

formation; innate immunity induction;22 and is useful in synthesizing some important vitamins (B complex and K). Large intestine bacteria are responsible for fermenting indigestible carbohydrates. A major consequence to this fermentation is the release of acids and gases such a carbon dioxide, hydrogen, nitrogen, and methane. Some of these gases are odorless, but dimethyl sulfide is extremely odorous. On average, the large intestine produces about 500ml of gas a day and this number increases with high carbohydrate rich foods (eg, beans).19

Blood Flow

Gastrointestinal blood supply comes from hepatic, splenic, and left gastric branches

of the celiac trunk. Under normal resting conditions, the GI tract receives approximately

20-25% of cardiac output, increasing with meal consumption and decreasing with intense

exercise.19,23 In addition to food and exercise, other factors such as age, temperature

(environment or body), training status, and disease affect GI tract blood flow.23 In general,

blood flow is regulated by the autonomic nervous system’s parasympathetic and

sympathetic divisions.19

Parasympathetic Nervous Division

Parasympathetic nerve fibers originate from the cranial and sacral spine.19 The

vagus nerve accounts for approximately 80-90% of cranial parasympathetic fibers, innervating the heart, lungs, and majority of visceral organs, including the GI tract (liver,

gallbladder, stomach, pancreas, small intestine, and proximal large intestine).1,19 At rest

and during digestion, the parasympathetic nervous division slows HR and respiration and

dilates blood vessels to the GI tract to allow relaxation and stimulate food digestion.1 At

onset of exercise, and during low to moderate intensity exercise, parasympathetic nerves

are inhibited to allow HR to increase. When an individual approaches maximum intensity

exercise, parasympathetic nerves are further inhibited and sympathetic nerves are directly

stimulated.1

Sympathetic Nervous Division

Much more complex, the sympathetic nervous division is commonly referred to as

the “fight or flight” response. Sympathetic fibers originate in the thoracic and lumbar spine

and innervate the heart, lungs, all visceral organs, all arteries and veins, and skin.19 The

sympathetic division stimulates release of norepinephrine and epinephrine to increase HR,

BP, and respiration,1 and vasoconstrict cutaneous and visceral blood vessels to shunt blood

to working muscles and limit nonessential functions (eg, digestion).19 During extreme

intense exercise, visceral blood flow may decrease up to 80%,24 placing these organs at risk for ischemic injury.

Other Regulators of Blood Flow

The greatest control of blood flow during exercise comes from central command, where the cerebral cortex controls anticipatory responses to exercise as well as responses during activity through the medulla and sympathetic-parasympathetic nerves. Baroreceptor and mechanoreceptors, located in blood vessels, respond to changes in mechanical pressure. Muscle chemoreceptors respond to changes in oxygen, carbon dioxide, hydrogen levels, and other metabolites from muscle activity. Other responses include vasoconstriction of blood vessels in inactive muscles and GI blood vessels and vasodilation in active muscles. Central command responses are faster than sympathetic responses

(sympathetic stimulation typically does not take place until moderate to intense exercise) and are involved with emotional responses to exercise or stress.1

Other hormonal regulators of blood flow include angiotensin II, aldosterone, vasopressin, and atrial natriuretic peptide. Often working collectively, these hormones exert their effects by either acting on the heart, blood vessels, or kidneys. The renin- angiotensin-aldosterone mechanism occurs in response to sympathetic nerve activity.

During hypovolemia or hypotension, renin, an enzyme released by juxtaglomerular cells, promotes production of angiotensin I. Angiotensin-converting enzyme converts angiotensin I to angiotensin II, causing vasoconstriction and subsequent increase in BP.

Also stimulated is aldosterone secretion from the adrenal cortex. Aldosterone stimulates reabsorption of sodium in the kidneys, subsequently pulling water with it and increasing both blood volume and pressure.1 Vasopressin is a hormone released by the posterior

pituitary in response to hypovolemia, hypotension, or increased plasma osmolality (ie, high plasma sodium concentration). In order to reestablish vascular homeostasis, vasopressin acts on the kidneys to promote water reabsorption. Thus, it is alternatively known as anti- diuretic hormone.1,25 Lastly, atrial natriuretic hormone responds to atrial stretching, often

from increased BP. Acting as a protective response and “fine tuning” BP, atrial natriuretic

hormone inhibits vasopressin, aldosterone, renin, and sodium reabsorption in the

kidneys.1,25

Gastrointestinal Barrier

Overall purpose of the GI barrier is to prevent foreign substances such as food product, acids, and toxins from passing from the intestine into the blood stream.26 Barrier

maintenance is imperative, and its function is tightly controlled. The GI barrier is

comprised of 3 factors: physical, physiological, and immunological. Physical factors

include the 4 epithelial tissue membranes layers. Membrane tissue integrity is primarily

maintained by the apical junctional complex.19,27 Together, physiological factors (ie,

mucus secretions and bicarbonate) and immunological factors (ie, macrophages) protect

the tissues and entire GI tract from toxins and acid.19,28

Tissue Membranes

Mucosa is the innermost layer of moist epithelial membrane that lines the lumen.

Major mucosa functions include secretion of mucus, digestive enzymes, and hormones

(making the mucosa an endocrine organ); absorption of digested end-products into the

blood stream; and protection against infectious disease.19 Stomach mucosa is exposed to

extremely harsh gastric juice.19 The acidity of gastric acid is imperative in defense against

excessive bacteria formation.18 However, exposure to gastric acid can cause severe

epithelial or organ tissue damage.21 Therefore, mucosa cells, which are rich in bicarbonate,

are imperative to protect and neutralize pH at the epithelial cell surface. Any damaged cells

are quickly shed and replaced by undifferential stem cells residing in gastric pits. The entire

epithelium surface is completely replaced every 3-6 days.19,21

The mucosa has 3 sublayers: lining epithelium, lamina propria, and muscularis

mucosae.19 Lining epithelium is heavy in mucus secreting cells and biocarbonate alkaline

secretions that create the protective GI barrier and allow food to pass through easier.19,21

Lamina propria, made up of loose areolar tissue, allows capillaries to reach the epithelial

layer and lymph nodes to fight against bacteria and pathogens.19 Muscularis mucosae is a

layer of smooth muscle cells that allow local movement of the mucosa.19

The second tissue membrane layer, the submucosa, is made up of elastic connective tissue that allows the stomach to expand and regain its normal shape after a large meal.

This layer contains blood vessels, lymphatic vessels, lymph nodes, and nerve fibers.19

Muscularis externa, the third tissue layer, is responsible for segmentation and peristalsis.

The smooth muscle contractions and relaxations propel contents through the tract and is

collectively known as GI motility. The circular layer creates sphincters throughout the tract

to prevent backflow and control food passing from one section to the next.19 The fourth,

outermost layer is the serosa, which is made up of areolar connective tissue covered by a

single layer of squamous epithelial cells.19

Apical Junctional Complex

In healthy GI barriers, epithelial cell integrity is maintained by the apical junctional complex, made up of tight junction, adherens junction, and desmosome.27 Tight junctions

are multi-protein complexes formed by distinct contacts between two adjacent plasma

100

membranes.29 Tight junctions contain transmembrane proteins, called claudins, and

peripheral proteins, called zonula occludens. Tight junctions are supported by adherens

junctions and desmosomes. Adherens junctions provide strong support to membranes to

allow cell-cell communication.27 Both tight and adherens junctions are connected to a

perijunctional actomyosin ring,29,30 which controls junction position. When actomyosin is

relaxed tension is reduced on the junctions, decreasing tight junction permeability. On the

other hand, when actomyosin is contracted, junctions are moved, and tight junction

permeability increases.30 Movement of tight junctions is driven by myosin light chain kinase, which phosphorylates myosin light chain in the perijunctional actomyosin ring.31,32

Tight junctions vary along the entire GI tract, differing in their tightening ability

depending on the segment function. For example, duodenum tight junctions have greater

ability to tighten than those in the ilieum.33 This is important because the duodenum is

exposed to more acid chyme leaving the stomach than the ilieum. Compared to the small

intestine, the colon has greater ability to tighten.33 This is likely because most nutrient

absorption has already occurred and cells need to be protected from the large bacterial

colony.

Gastrointestinal Distress

Gastrointestinal distress can present as any number of symptoms, including

abdominal pain, cramping, bleeding, dyspepsia, constipation, diarrhea, nausea, emesis,

reflux, or urge to urinate or defecate.34,35 Unfortunately, etiology of these symptoms is often

complex and sometimes not well understood due to a number of confounding factors within

and between subject populations. Gastrointestinal distress can occur from systemic

conditions or from GI damage/irritation. Systemic driven GI distress may originate from

101

triggers such as stress, emotions, fever, fatigue, or other diseases.34,35 Direct damage or

irritation can be caused by GI disease, trauma, or even diet.36

Etiology of GI Distress Symptoms

Abdominal Pain

Abdominal pain may originate from referred, mechanical, inflammatory, or

ischemia-reperfusion sources. Conditions such as muscular injuries, muscle guarding, or

spinal pathologies may cause referred abdominal pain.34 Mechanical abdominal pain may

occur from stretching of an organ wall, jarring or vibrations, increased abdominal pressure,

body position, or contraction of abdominal wall and diaphragm.35,37,38 Inflammatory pain

originates from release of inflammatory mediators.39

One of the primary causes of abdominal pain is GI ischemia. Appropriate blood

flow is not only important in providing oxygen and nutrients to tissues,19 but in buffering

acid exposure and healing damaged tissue.40 Ischemic pain occurs when blood flow to an

area is decreased to the point where tissues are not supplied with appropriate oxygen and

nutrients.34 Vascular endothelial cells are particularly vulnerable to ischemia, presenting

with altered membrane potential, decreased fluidity, impaired cytoskeleton, decreased

energy stores, or releasing inflammatory mediators.41 Reperfusion following ischemia has

also been identified as a source of pain and inflammation, causing swelling, disruption of

the basal membrane, leukocyte adherence, and production of reactive oxygen species

(ROS).41,42

Gastritis and Peptic Ulcers

General stomach wall inflammation is referred to as gastritis. Gastritis includes a

number of conditions that typically only affect the mucosal layer and is classified as acute

102

or chronic. Acute gastritis includes mucosal lining erosion. Causes are sometimes unclear

but often associated with medication, alcohol, or stress. Chronic gastritis is less common

and includes causes such as diabetes, autoimmune diseases, or infection by helicobacter

pylori bacteria.19,21,34 Signs and symptoms of gastritis vary, some individuals are

asymptomatic, but most common symptoms include abdominal pain or discomfort, nausea,

vomiting, heartburn, or occult blood loss.34

An ulcer occurs when erosion reaches the submucosa layer. Duodenal ulcers are

more common than gastric ulcers, and, like gastritis, are most commonly caused by

bacteria. Gastric ulcers are most commonly caused by NSAIDs. Other causes include

alcohol, coffee, smoking, trauma (eg, burns), and increased age.19,34 Psychological stress

is also a contributing factor, especially in individuals with recurrent ulcers.34 Both gastric

and duodenal ulcers present similarly, with severe pain, burning, cramping or aching,

distention, and nausea. Pain typically fluctuates with gastric secretions and food intake. In

duodenal ulcers, pain typically occurs 1-3 hours after eating. On the other hand, eating may either relieve or exacerbate gastric ulcers.34 This is because presence of food allows gastric acid to target something other than the stomach lining; however, food could be the irritant causing damage. If left untreated, ulcers could lead to perforation of the stomach or intestinal wall, peritonitis, and/or severe hemorrhaging.34

Nausea and Emesis

Nausea is stimulated from the autonomic nervous system when stomach nerve

endings are irritated. Causes of nausea can range from strong emotions to toxins such as

drugs or alcohol that irritate the GI tract.34 Emesis can occur from a number of triggers,

including bacterial irritation, alcohol, and certain drugs. The medulla receives an impulse

103

from irritated sites that triggers abdominal wall and diaphragm contraction. Intra- abdominal pressure increases, then the cardiac sphincter closes, soft palate rises to close off the nasal passage, and contents from the stomach, and potentially duodenum, are forced upward.19

Dyspepsia and Gastrointestinal Reflux

Dyspepsia is general abdominal discomfort, sometimes referred to as indigestion.

Dyspepsia is often caused by gastroesophageal reflux. When stomach contents move backward into the esophagus the acidity causes a burning sensation, usually located behind the sternum (heartburn). Some causes of gastroesophageal reflux include alterations in esophageal peristaltic activity; increased gastroesophageal sphincter relaxation; consumption of alcohol, spicy or highly seasoned foods; medications; stress or nervousness; and large movement (eg, bending and lifting) after a large meal.34,43,44

Diarrhea and Constipation

Diarrhea and constipation occur when food product passes too quickly or too slowly

through the large intestine.19 If stool passes quickly water is not absorbed and stool becomes “loose”. When stool sits too long in the large intestine too much water is absorbed and stool becomes hard. Specific causes of diarrhea and constipation vary, but include disease, diet, medications, and strenuous exercise. Constipation can also cause mechanical abdominal pain.34

GI Bleeding

Depending on the cause, GI bleeding can be identified by either brown colored

emesis, hematemesis, melena (black, tarry stool), or hematochezia (maroon colored

stool).34 Severe GI bleeding during exercise is rare, but may be associated with peptic

104

ulcers, trauma, systemic illness, or chronic alcohol or NSAID use.34 Most often GI bleeding presents as occult blood in feces and occurs from stomach damage.35 Symptoms such as

nausea, vomiting, diarrhea, and abdominal pain may be secondary to GI bleeding.34

Measurement Techniques

Gastrointestinal Distress Questionnaires

Qualitative surveys are inherently limited due to subjectivity, but are clinically

useful tools to determine how someone feels. Gastrointestinal symptoms are commonly

assessed prior to and after physical activity in order to determine change in GI symptoms

that may be attributed to exercise or experimental conditions.36,45,46 Administering

questionnaires during physical activity may be done,46-49 although this is more practical during laboratory studies.

There are number of other challenges to existing GI symptom research. A common limitation is survey variability across studies. Some questionnaires provide specific questions to distinguish upper and lower abdominal symptoms, number of occurrences and severity,36,43 but others are less descriptive. Not identifying GI symptoms at rest (baseline) and not controlling for diet, medications, or supplement use is another common limitation.

Lastly, timing of survey administration also varies, with post-activity time ranging from immediately45 to 2 hrs post.36

Endoscopy

Endoscopy is the gold standard to objectively quantify GI damage.50 Sometimes

combined with biopsies, endoscopy allows medical providers or researchers to examine

the GI lining, identify specific damage, and measure severity. Endoscopy is frequently

used, but invasive and requires intensive equipment.

105

Occult Fecal Blood Measures

Another objective measure of GI damage is occult fecal blood. There are two major

types of fecal tests – guaiac-based fecal occult blood test (g-FOBTs) and fecal immunological test (FIT). Each has unique advantages and disadvantages. Regardless of test type, kits can be self-administered and are recommended for early detection of serious

GI diseases.51

The g-FOBT uses guaiac to test for heme in stool. Differences exist among test

brands, some with lower sensitivity for occult blood than others.50 Advantages to g-FOBT are its specificity and ability to detect blood loss from any portion of the GI tract.51 A major

disadvantage of g-FOBT is interference with certain plants and red meat. Red meat contains heme, which may alter tests results. Individuals using g-FOBT are instructed to

not eat certain foods for at least 72 hrs prior to conducting the test.52

The FIT was introduced as a more selective alternative to g-FOBT. Designed to use anti-bodies to selectively detect globin, FIT identifies colon blood loss. By detecting globin rather than heme, there is no dietary interference or potential drug interactions. The major disadvantage to FIT is inability to identify small intestine or stomach bleeding.51

Since fecal occult tests are commonly used for early detection of cancer, specificity

and sensitivity compared to colonoscopy is of particularly high interest. The FIT has been

shown to have significantly higher sensitivity compared to g-FOBT in detecting cancer,50

with 65-90% sensitivity with one-time testing.51 On the other hand, g-FOBT has significantly higher specificity.50 Considering advantages and research outcomes, FIT is

the recommended method for detecting occult fecal blood.51

106

Gastrointestinal Permeability

The apical junction complex, specifically tight junctions, closely regulates GI

permeability. There are 2 primary permeability pathways: paracellular and transcellular.

Paracellular permeability allows passive diffusion of large molecules, like proteins and

bacteria, between adjacent cell membranes. Transcellular permeability allows active or

passive diffusion of small molecules, such as ions, through specific cell membrane

channels.27,53

The GI tract has a usual amount of permeability for small molecules (< 150 Da)

through non-mediated diffusion.53 Gastrointestinal permeability is considered leaky

because it naturally allows a certain amount of fluid and ion exchange across the surface.27

On average, 8-9 L of fluid pass through the GI tract daily. Of that, only 100-200 ml is not

absorbed and excreted through urine and feces. In the event of increased permeability,

decreased fluid reabsorption can present clinically as diarrhea.54 A number of other molecules can improperly pass through the GI barrier. The immune system can typically neutralize escaped molecules unless there is a state of severe stress or disease. For example, if LPS molecules in an immunocompromised individual are allowed to pass from the GI tract to the bloodstream, local and systemic inflammation can ensue and further increase permeability. If uncontrolled, serious conditions like endotoxemia, septic shock, EHS, or death can occur.26,29

Tight Junctions

Tight junctions are the rate-limiting step in regulating paracellular permeability.29

Tight junctions are dynamic, continuously undergoing remodeling and restructuring as

they interact with outside stimuli. Inflammatory mediators, hormones, and pathogens

107

increase permeability at tight junctions through two mechanisms: 1) actin-myosin ring dysfunction through cytoskeleton disruption, and 2) direct damage to tight junction proteins.54 Specific mechanisms include stimulated myosin light chain kinase transcription,

endocytosis or transmembrane protein cleavage, altered protein kinases and second

messengers, and increased claudin expression.27

Research on chronic inflammatory bowel diseases and cell cultures have associated

tight junction dysfunction to elevated inflammatory cytokines and inflammatory

enzymes.55-57 Pro-inflammatory cytokines alter intestinal permeability by increasing tight

junction protein transcription, protein degredation, and modulating kinases and

cytoskeleton.27 Others have been shown increased permeability by stimulating enzymes that cleave phospholipid membranes57 and epithelial-neutrophil interactions.58

Endotoxin

As previously mentioned, there is an extensive bacteria flora throughout the GI tract. The outer cell membrane wall of gram-negative bacteria is predominately made up

of LPS. Unlike gram-positive bacteria, the complex membrane is designed to protect

against outside substances, making them more resistant to immune responses and antibiotic

medications.59 Three components make up LPS, an O-polysaccharide chain (O-antigen), a core oligosaccharide sequence, and a lipid A region. The lipid A portion contains hydrophobic fatty acid chains and is toxic in humans. Therefore, LPS is often referred to as endotoxin60 and entry into the circulation as endotoxemia.

Under healthy conditions the GI barrier prevents LPS from entering the circulatory

system. A certain amount of LPS in blood is normal (< 0.10 ng/ml),61 coming from dead

bacteria and LPS shedding.62 The liver and immune system work together to clear LPS.63

108

As blood filters through the liver Kupffer cells and hepatocytes neutralize LPS through

endocytosis.64 A major immunological response comes from neutrophils releasing

bactericidal/permeability-increasing proteins. They have a high affinity for endotoxin65 and

neutralize activity by decreasing membrane integrity, altering electrochemical gradients,

inducing apoptosis, and inhibiting cytokine release.66,67

Measurement of GI Permeability

Compared to animal models, there are few techniques for measuring GI

permeability in humans. Measures of plasma LPS provide a general indicator of GI

permeability,28 but have inherent limitations during analysis and cannot provide specific information regarding extent and location of GI permeability. Probes such as sugars/carbohydrates, chromium-labelled ethylenediaminetetra-acetate (Cr-EDTA), and

polyethylene glycol (PEG) are commonly used to determine altered permeability

location.28,68

Sugar/Carbohydrate Probes

A common permeability technique utilizes urine measures of non-digestible, non-

metabolized probes in an orally ingested solution.28 Solutions typically include 3 sugars:

sucrose, lactulose, and rhamnose;28 however, others such as mannitol, sucralose, and

xylose have been used.26,69 Sucrose (molecular weight = 342 Da) is not digestible in the

stomach, and under normal healthy conditions will pass into the small and large intestine

where it can be absorbed.69 When stomach permeability increases it allows sucrose to leak out. Therefore, presence of sucrose in the urine indicates increased stomach permeability.

Similarly, lactulose is not digestible in the small intestine. Due to its large molecular weight

(342 Da) lactulose only passes through paracellular routes (ie, tight junctions) and its

109

presence in urine indicates increased small intestine permeability.28 To control for non-

barrier (GI permeability) related urinary excretion, such as GI transit time, fluid

distribution, and renal clearance, rhamnose (molecular weight = 164 Da) is taken with

lactulose.26,28 Together, rhamnose and lactulose should be affected similarly by any non- barrier related factors. The difference between rhamnose and lactulose is attributed to absorption route, where rhamnose detects “small pore” (para- and/or transcellular) permeability and lactulose detects “large pore” (transcellular) permeability.26,28,68 Intestinal permeability is presented as a percentage, or ratio, of lactulose to rhamnose in urine.26,28

Drawbacks vary depending on type of sugar used. Lactulose and other sugars are present in some foods, which may be excreted in urine. Sugar probes are susceptible to metabolism and degradation from bacteria, which would underestimate permeability.68

However, research has shown no significant difference in intravenous delivered lactulose

recovery (92.7 + 1.2%) compared to Cr-EDTA (97.4 + 0.5%), which cannot be metabolized

and is not degraded by bacteria.68 This suggests lactulose is an appropriate measure for GI

permeability. Overall risk of consuming the solution is low, but some reported side effects

have included minor GI disturbances.53 Low-risk in combination with efficiency makes sugar probes the standard and preferred GI permeability technique.

Chromium-labelled Ethylenediaminetetra-acetate (Cr-EDTA)

A less commonly utilized technique, Cr-EDTA uses a γ-ray emitting isotope in an orally ingested solution. Percentage excreted in urine indicates more small intestine permeability. Advantages to Cr-EDTA is resistance to bacterial degradation.68 The major

negative effect of Cr-EDTA is the small exposure to radiation, albeit minimal compared to

an abdominal x-ray (0.12 mSv versus 1.4 mSv).69

110

Polyethylene Glycol (PEG)

A mixture of polymers, PEG comes in a variety of molecular weights (400-

4,000Da)28 that are orally ingested and measured for urinary concentration to determine

permeability. Specificity of PEG is high due to using polymers of varying masses to mimic

the mass of small molecules (eg, rhamnose), medium molecules (eg, lactuose and Cr-

EDTA), and large molecules (eg, LPS). Despite high specificity, use of PEG has declined

due to variability and inconsistency in GI measures. Using intravenous injected probes and

comparing to Cr-EDTA, percent recovery of PEG-400 polymers was only 40.7 + 1.8%.68

Losses are attributed to PEG’s lipid solubility, allowing it to more easily pass through

tissues and, in the case of intravenously injected probes, pass into the GI tract.70,71 Large

losses of PEG polymers lead to underestimated permeability, making it less sensitive in

68 detecting small permeability changes.

Plasma Lipopolysaccharide

Plasma LPS concentration provides a general idea of GI barrier dysfunction and is

frequently used in human research to measure endotoxemia.61,72-74 Limulus amebocyte

lysate assays are the preferred technique. They are highly sensitive and measure biological

endotoxin activity.75 Unfortunately, there are several disadvantages when using this

measure. First, plasma LPS levels do not identify the specific area of GI barrier dysfunction

(ie, gastroduodenal, small intestine, or colon). Second, because LPS is located in the

external environment, there is a risk for assay contamination during collection, handling,

and storage. Third, and not well understood, LPS can be bound or cleared by a number of

biological components (ie, platelets, bile salts, high-density lipoproteins, and LPS-binding

111

protein). Inactivation is maximum when temperatures range 37-45°C, and could lower LPS levels below the assay’s detection ability.75

INFLAMMATORY RESPONSES

Whether caused by trauma or a pathogen the body will launch an immune response to neutralize the problem. Resultant signs and symptoms such as fever, pain, lethargy, swelling, and redness are often considered negative. In actuality, inflammatory responses are vital to prevent and counter threats. Unfortunately, inflammation can be negative if not controlled (eg, autoimmune diseases and secondary hypoxic injury). Therefore, many components of the inflammatory response have both pro- and anti-inflammatory roles depending on stimuli and these responses are closely regulated in attempt to maintain homeostasis.

The two types of immunity are acquired and innate. Acquired immunity includes specific responses that have adapted due to previous exposure. These responses include humoral and cell-mediated events (antibody secretion and proliferation of antigen-specific

T and B cells). Innate immunity includes all immune response that have no immunologic memory to previous pathogens.34 Innate responses include phagocytes (neutrophils,

monocytes, macrophages, mast cells, and tissue dendrite cells) and basophils, eosinophils,

and lymphocytes.19,34 Often working congruently with innate and required responses, other

components vital during inflammation include cytokines, heat shock proteins (HSPs),

ROS, and prostanoids.

Inflammatory Components

Lymphocytes

112

Lymphocytes play a major role in inflammatory mediation and work in close

conjunction with antigen-presenting cells (APCs), such as phagocytes. After a phagocyte

digests a pathogen, antigenic material is presented on the surface so it can be recognized

more easily as a pathogen. Phagocytes then present the cell to lymphocytes, specifically

T4 lymphocytes (cells). T cells provide cell-mediated immunity and make up between 65-

85% of bloodborne lymphocytes.19 There are multiple subdivisions of T cells, two that are responsible for destroying pathogenic cells are T4 cells (helper T cells) and T8 cells

(cytotoxic T cells).19

Once a T cell is presented and/or bound to an antigen it must be stimulated to

continue with cell destruction or abort. This is referred to as costimulation. While there are

numerous types of signals, the major signal for continued destruction comes from

cytokines. Making up 75% of all T cells,34 T4 cells are vital to the immune response even

though they do not directly destroy a pathogenic cell. They “help” by providing

costimulation signals for destruction, activating macrophages, assisting natural killer cells

(NKCs), and stimulating proliferation of other T and/or B cells. Proliferation temporarily

increases activated T and B cells to respond to the current pathogen attack. It also

contributes to acquired immunity by forming memory T and B cells that remain in the body

until the same antigen is presented again.19,34 T4 cells are further classified as either Th1

or Th2, where Th1 cells mediate aggressive cytotoxic, phagocytic inflammatory responses

and Th2 cells mediate less-tissue destructive, cell mediated responses.76

Cytotoxic T cells directly attack and destroy a cell. These cells bind to a presented

antigen and, depending on the type of T8 cell, release a cytotoxic chemical into the target

cell’s plasma membrane. The T8 cell then detaches from the target cell and continues

113

throughout the body looking for other pathogenic cells. Cytotoxic chemicals typically

accompany perforin, a cytolitic protein that forms transmembrane pores in target cells.19

Once a pore is formed, chemicals like granzymes, a family of serine proteases, or TNFs

enter and trigger apoptosis by cell lysis and/or DNA fragmentation.77,19

Regulatory T (Treg) cells are a subset of T cells that are less understood. Known to

be immunosuppressive, these cells are designed to slow down or stop immune responses

by releasing anti-inflammatory cytokines,19 inhibiting T cell activation and cytokine

release, inhibiting APCs,78 and upregulating Treg cell production.79 Research has shown

Treg cells also contain cytotoxic properties, utilizing perforin80 and granzymes to control

T cells, NKCs, and other cytotoxic cells.79 While Treg cells are considered important in

preventing autoimmune reactions,19 excessive Treg production is implicated in tumor and

disease progression.81

Another type of lymphocyte, NKCs are important in initial immune response and are less specific than adaptive immune lymphocytes. As a result, NKCs are able to target many different viruses, bacteria, and tumor cells with or without an APC.19,34 Their primary function is to destroy foreign or damaged cells; this is predominately done by targeting cell membranes and releasing perforin to create cell lysis.82 In addition to direct cell destruction,

NKCs promote cell death by influencing T and B cell response and producing a variety of cytokines and growth factors.83

Cytokines

Cytokines are chemical mediators, connecting the immune system to other body systems. Cytokines regulate responses like cell proliferation, apoptosis, chemotaxis,19,34,59

fever, leukocytosis, acute protein synthesis, muscle catabolism, and the hypothalamic-

114

pituitary-adrenal axis.84 There are several types of cytokines; interleukins (ILs) and TNFs

are two families.

Interleukin (IL)

There are more than 30 different ILs found in humans, identified numerically and

often with additional sub-classifications. Originally identified as chemical mediators

produced by leukocytes (“-leukin”), in actuality T4 cells, NKCs, macrophages, endothelial

cells, monocytes, and other cell types express ILs. Interleukins act as either pro- or anti- inflammatory mediators by modulating growth, differentiation, and activation.85,86

The pro-inflammatory cytokine IL-1 is primarily released by macrophages and

stimulated by other cytokines (IL-4 and IL-6) and endotoxin.86 One major function of IL-

1 is to co-stimulate T cells to release IL-2, a growth factor that activates NKCs and

enhances proliferation of B cells, T cells, and macrophages.19,86 Other IL-1 functions are

fever induction by increasing the hypothalamus’ set-point temperature,19,34 stimulating PG

production,34 hypotension during septic shock, and inducing sleep and lethargy.86

A prominent pro-inflammatory mediator during early immune responses,85 IL-6 is

primarily induced by monocytes and other cytokines (IL-1 and TNFs).87 IL-6 is

multifunctional, enhancing T cell proliferation and activity, B cell differentiation and

antibody production, macrophage differentiation, and positive acute-phase protein

synthesis.86,87 IL-6 also acts as a thrombopoietin, promoting platelet production,87 and,

along with IL-1, is responsible for fever and IL-2 expression.86

An anti-inflammatory mediator, IL-10 is primarily expressed by monocytes, T cells, and B cells.88 IL-10 is effective at inhibiting APCs89 and T4 cell, phagocyte, and

NKC mediated expression of IL-1β, IL-6, and TNF-α.85,86 The IL-10 family includes IL-

115

22, which is prominent in anti-apoptosis and cell repair in inflammatory skin, liver, and GI

diseases.90 IL-22 is suggested to protect GI barrier integrity by promoting cell proliferation and increasing expression of anti-microbial molecules and mucins.90

Tumor Necrosis Factor (TNF)

The most prominent TNF member is the pro-inflammatory cytokine TNF-α, also

referred to as cachectin. These cytokines are derived from neutrophils, phagocytes, T cells,

endothelial cells, and mast cells.55,86 TNF-β is primarily produced from lymphocytes86 and has historically been referred to as lymphotoxin. TNFs have direct cytotoxic effects and promote destruction of target cells, particularly tumor cells, by activating T cells and

phagocytes.19,86

Research has identified a clear relationship between TNF-α and severe cachexia in

chronic infections and cancer.86 Along with IL-1, TNF-α contributes to hemorrhagic necrosis,87 vascular endothelial cell lysis,91 and stimulates IL-6 expression.87 TNF-α is

highly induced by endotoxin,92 causing increased tight junction permeability by

stimulating myosin light chain kinase27 and increasing vascular permeability. As a result,

TNF-α is a key mediator in septic shock.92

Heat Shock Protein (HSP)

Heat shock proteins are molecular chaperones that assist in proper folding during

protein synthesis.60 There are many different HSP families numbered according to their

molecular weight.19 Majority of HSPs are constitutive60 and found abundant throughout

the body.93 A few HSPs are inducible, protecting cells exposed to stressors such as

hyperthermia, hypothermia, inflammation, trauma, energy depletion, hypoxia, or

toxins.19,94,95,96

116

Understanding exact HSP mechanisms is complicated, particularly since they have

many functions and protect different cell areas. One suggested induction mechanism is in

response to stress signals from abnormally folded and denatured proteins.97 Other research suggests HSPs are induced by increased reliance on anaerobic metabolism following mitochondria damage98 or altered cytoskeleton structure resulting in inhibited

mitochondria function.99 Still yet, another mechanism is in response to inhibited RNA

processing following cell nuclei thermal stress.100 Regardless of the exact mechanism, HSP induction is associated with an increased demand for protein synthesis.

Primary functions of HSPs are to prevent denaturation and/or allow damaged cells to undergo apoptosis rather than necrosis by maintaining mitochondrial function.101

Apoptosis is a controlled event and requires ATP. If mitochondria are damaged and cells

undergo necrosis, leaked cell contents may perpetuate or trigger inflammatory

responses.101 More recent research suggests HSPs have a variety of pro- and anti-

inflammatory functions during immune responses. HSPs may facilitate APCs,102 improve

tolerance to103,104 and inhibit expression of TNF-α and IL-1,105,106 and promote NKC

activity.96

Another protective function of HSPs is development of thermotolerance. Increased

expression and accumulation of HSPs following thermal stress allows cells to survive

subsequent stress by protecting against protein denaturation. Thermotolerance is

temporary, lasting 3-5 days, and dependent on initial temperature and duration of

exposure.107 Considered one of the most highly inducible,108 HSP70 is preferentially induced during thermal stress and protects against ROS and cytokine driven apoptosis.94,101,109,110

117

Reactive Oxygen Species (ROS)

Reactive oxygen species are free radicals (eg, superoxide, hydrogen peroxide, and

hydroxyl) released as byproducts of cell and body processes (eg, aerobic metabolism).101

These free radicals are extremely reactive to cells60 and easily pull electrons from

molecules.59 Several mechanisms increase ROS production. During hypoxia the imbalance

between inputting electrons for fuel oxidation and transferring electrons to molecular

oxygen results in ROS formation.60 Other mechanisms for ROS release are ischemia-

reperfusion injury,42 phagocytosis,101 and arachidonic acid metabolism.111

Balance between positive and negative ROS effects is complex and depends on

other inflammatory responses. A primary function of ROS is regulation of stress

proteins.101 Acting like second-messengers, they trigger activation of other pathways and factors.112 Some ROSs are cytoprotective, upregulating HSPs by causing a cell to express

a stress response or inducing some protein denaturation.101,112 Other ROSs provide defense

against pathogens and toxins.101

Despite their protective anti-inflammatory effects, ROS also promotes pro-

inflammatory responses by creating oxidative stress and cell damage. A primary target for

ROS damage is lipid membranes. For example, ROSs stimulate phospholipase A2,111

which breaks down phospholipid membranes by hydrolyzing fatty acids. ROSs have also been shown to increase cytokine gene expression.111 Excessive ROS accumulation can

result in cytotoxic effects to enzymes, amino acids, DNA, and mitochondria.60,112

Cyclooxygenase (COX)

Trauma and other inflammatory stimuli activate phospholipase A2,60 which releases arachidonic acid, a crucial fatty acid incorporated in the phospholipid membrane

118

structure. Arachidonic acid is the precursor for production of 3 eicosanoids: prostaglandin

(PG), thromboxane (TX), and leukotriene.60 Leukotriene production is catalyzed by

lipooxyngease, which is not inhibited by NSAIDs113 and therefore will not be further

discussed in this literature review. To produce TXs and PGs (collectively known as

prostanoids), arachidonic acid must be converted to the prostaglandin endoperoxides PGG2

114 and PGH2. These conversions are catalyzed by PGH synthases, more commonly

referred to as cyclooxygenase (COX).115

There are two well established COX derivatives in humans, COX-1 and COX-2.

Both derivatives are responsible for converting arachidonic acid to TXs and PGs,116 but

vary in their functions. Inherently located in most tissues, COX-1 is responsible for

production of prostanoids that regulate gastric mucus secretions, platelet aggregation, and

renal blood flow.25,116 Present in small amounts naturally, COX-2 is induced locally by inflammatory mediators (eg, cytokines and endotoxin)115,76 and mediates inflammation,

pain, and fever.25,60,116

Prostanoids

All prostanoids are derived from prostanoic acid, and most commonly by

arachidonic acid. Prostaglandins possess a 5-member carbon ring, with different types

designated by letters A-K in accordance with the nature and position of substituents.117 In

humans, the most common biologically active PGs are dienoic (designated by a subscript

118 60 2). These PGs include PGE2, PGD2, PGF2, PGI2 (prostacyclin), and PGA2 and PGJ2

(cyclopentenones).115

In general, PGs are essential to maintaining GI mucosal barrier integrity, vascular

tone, and renal function. They also are responsible for pain, inflammation, and implicated

119

in certain chronic diseases. Hormonal stimuli, such as angiotensin II,119 vasopressin,120 or

bradykinin,121 is typically necessary for most PG synthesis, but some PGs can be induced

by cell-cell interactions.117 In order to exert their effects, PGs must be actively transported

across membranes. However, because they are readily deactivated, they exert only local

effects.117 Non-cyclopentenone PGs act through binding to specific transmembrane G protein receptors. For example, PGE2 receptors are designated E prostanoid receptor (EP)

1-4. Once bound and depending on the desired action, effects may include up or down

regulation of second messengers cyclic adenosine monophosphate (cAMP) or calcium,

altered membrane potential, or activation of specific protein kinases.117,122

Identifiable by an oxygen bridge between carbon 6 and 9,117 PGI is important in

maintaining cardiovascular function.122 Endothelial and vascular smooth muscles cells

constitutively express PGI.117 Prostacylin is a potent vasodilator and inhibitor of platelet

aggregation, leukocyte adhesion, and vascular proliferation.114,123 Its efficacy led to

development of PGI as a pharmacological treatment in a number of cardiovascular and

renal diseases.114

Derived from PGE and PGD respectively, PGA and PGJ contain a unique

cyclopentenone ring structure. Cyclopentenone PGs contain an α,β–unsaturated carbonyl

group that can react with glutathione (GSH) or other target cell proteins.117 Glutathione is

an anti-oxidant that protects cells against ROS and other toxins. Cyclopentenone PGs are

highly active; by reacting with GSH, they elicit a number of physiological effects other

PGs cannot. For example, PGA and PGJ have been shown to induce HSP74 by inhibiting cell proliferation.124 They can also induce apoptosis in tumor cells125 and inhibit DNA and

RNA viruses.126

120

Instead of a 5 carbon ring, TXs have a heterocyclic ring.117 Thromboxanes with

unstable bicyclic oxygenated rings are designated TXA, and those with stable oxane rings

TXB.117 Like PGs, subscript numerals distinguish the number of double bonds117 and they exert their effects through G protein receptors.122 All prostanoids have relatively short half-

lives. However, TXs are extremely unstable, with 20-30 sec half-lives.117,122 In comparison,

under normal blood pH and temperature the half-life for PGI is 2-3 min.127 Though TXs

elicit inflammatory effects, TXA2 is predominately produced from COX-1 in platelets.

Along with PGIs, TXs play a major, albeit opposing, role in vascular function.117

Functions of Prostanoids

Gastric Mucosal Protection

Throughout the GI tract COX-1 is expressed constitutively and produces cytoprotective PGE2 and PGI2. Damage or irritation to gastric mucosa epithelial lining up-

128 regulates COX-2 expression, and subsequently PGE2 and PGI2 as a defensive, anti- inflammatory response.18 Research on the exact protective mechanisms has predominately

focused on animal and cell cultures. Additionally, like PGI analogues developed for

cardiovascular disease treatment, the effectiveness of PGE in protecting the GI mucosa has

led to use of PGE analogues in studies. Existing research concludes a multifaceted

protection by maintaining blood flow, inhibiting cell apoptosis, reducing acid secretion,

promoting mucus and bicarbonate secretion, inhibiting ROSs, inhibiting GI hypermotility,

and maintaining tight junctions.

Increased Epithelial Blood Flow

Vasodilation by PGE2 and PGI2 increases blood flow, which prevents cell necrosis

by increasing platelets and promoting angiogenesis and cell proliferation. In addition,

121

preserving adequate GI blood flow maintains relatively neutral pH even during times of

high acid secretion.18 Importance of blood flow is evident in GI diseases or conditions where blood flow is impaired. In patients with portal hypertension there is a higher risk of developing GI ulcers and bleeding. This risk is associated with decreased PG,18 which was

shown in rodents with reduced portal blood flow from stomach cirrhosis. Cirrhotic rodents

exposed to a mucosal irritant had a significantly blunted blood flow compared to controls

(2 + 8% vs 51 + 5%, P < .001). However, PGE administration significantly increased blood

flow in cirrhotic rodents compared to controls (83 + 12% vs. 22 + 8%, P < .01).129

Apoptosis Inhibition

Ability for PGs to prevent mucosal cell apoptosis has been shown in vitro. Isolated

guinea pig gastric mucosal cells placed under maturation-dependent stress were used to mimic high turnover rate seen in vivo with acid exposure and damage. When compared to control cells, PGE2 inhibited spontaneous cell apoptosis and significantly increased cell

viability (P < .01).130 Apoptosis inhibition occurred even after DNA fragmentation

occurred by protecting mitochondria in a rapid, yet transient and dose dependent manner.130

Similarly, when cells were stressed by 4% ethanol, PGE2 significantly increased cell viability (P < .01) compared to control cells.131

Inhibited Gastric Acid Secretion

Although somewhat controversial, PGI2 and PGE2 inhibit gastric acid secretion.

Healthy males orally administered PGE2 showed significantly reduced gastric acid

secretion by approximately 40% compared to control (P < .05).132 These results are supported by studies linking acid secretion and gastric ulcer development. A 12 week pharmacological study compared no treatment, a proton-pump inhibitor (known acid

122

suppresser), and a PGE1 analogue on GI ulcer development in 458 patients with previous

gastric ulcer history. After 12 weeks of NSAID use, 93% of patients using PGE were ulcer

free compared to only 51% of control and 81% of proton-pump inhibitor participant. Of

those who developed ulcers, time to occurrence was significantly prolonged with PGE (P

< .001) compared to no treatment and was not significantly different than the proton-pump

inhibitor.133

One suggested mechanism of gastric acid inhibition is increased mucosal blood

flow, but others believe PGs elicit non-blood flow mediated inhibition. Intravenous PGE2

and PGI2 in dogs significantly inhibited acid secretion (P < .05). PGI2 resulted in increased

134 mucus blood flow while PGE2 decreased blood flow. Even stronger support for PGE acid secretion inhibition comes from in vitro studies where blood flow is not a factor.135

This suggests PGE2 and PGI2 reduce acid secretion by directly inhibit mucus secretory cells. These mechanisms are not clearly understood, but include altered intracellular cAMP135,136 and changes in cell membrane bound phospholipids that alter hydrogen, potassium, and ATPase activity.137

Other studies have been unable to demonstrate significant reduction in acid

secretion.138,139 Lack of agreement may be attributed to different measurement techniques

or methods such as dose or duration of use. Type of PG used also explains conflicting

results. Unlike endogenous PGE2, intravenous or orally administered PGs analogues are

not rapidly metabolized and can exert longer lasting effects. This idea is supported by a

study in healthy males comparing PGE2 to different doses of PGE2 analogues. No

significant reduction in gastric acid secretion occurred with natural PGE2, but both

123

analogues resulted in significant decreases directly proportionate to dosage and lasting 3 hours after administration.140

Increased Bicarbonate and Mucus Secretions

Both in vivo and in vitro studies have demonstrated upregulating PGE2 production

increases bicarbonate and mucus secretions to protect the GI barrier. Canines administered

intravenous PGE2 resulted in significantly increased bicarbonate production compared to

control (mean = 64.5 μmol/hr vs 34.0 μmol/hr, P < .05).141 These results are similar to

studies on mucus secretion in amphibian cells,142 rabbit gastric epithelial cells,143 and

rodents.144 In 6 healthy humans administered varying doses of a PG analogue, mucus

secretion also significantly increased (P < .01) in direct proportion to administered

doses.138

Despite being abundant in gastric epithelium, research has been unable to identify endogenous PGI2 as a stimulator of bicarbonate secretion. One suggested reason for this is

the location of epithelial producing PGI2 cells. Unlike PGE2 cells, which are on the surface

near bicarbonate secreting cells, PGI2 cells are located deeper. Considering location and a

short half-life, PGI2 may not have enough time to elicit effects on bicarbonate cells, and

assumingly mucus cells, before being degraded.145

Decreased GI Motility, Neutrophil Chemotaxis, and ROS

Many protective mucosa effects are interrelated. This is particularly apparent when discussing GI hypermotility, neutrophil chemotaxis and ROSs inhibition. Hypermotility results in a number of GI disturbances and is associated with increased risk for lesions along mucosal folds.146,147 Repeated and excessive GI contraction and relaxation also leads

to microcirculatory disturbances, with consequences similar to ischemic-reperfusion

124

injury.146 Lastly, hypermotility stimulates neutrophil chemotaxis and neutrophil adherence

to vascular endothelium.148 When neutrophils increase at sites of inflammation they

interact and adhere to plasma membranes of endothelial cells and release ROS.149 If

neutrophils accumulate in microvasculature, blood vessel occlusion can potentiate

ischemic conditions.150

Exogenous PGs are potent inhibitors of hypermotility, neutrophils and ROS.146,151

Studies have demonstrated these effects using rodents with indomethacin induced GI

mucosa lesions. Compared to control, PGE2 significantly inhibited lesion occurrence by

83.6% (P < .05), and this was partly attributed to significant decreases in neutrophil chemotaxis and GI motility (83% and 81.3% respectively, P < .05).152 In a similar study,

results showed pre-treatment with PGE2 significantly inhibited lesion formation (90.6%,

P < .05) and GI motility.151 Using isolated neutrophils, release of ROS was significantly

inhibited by PGE2 (P < .05) and, like many other protective effects, was dose dependent.

This study also found PGD2, but not PGI2, was a significant and more potent inhibitor of

ROS release (P < .05).153 Ischemia and reperfusion are potent producers of ROS154 and decreases GSH availability,149 which counters ROS.

Decreased GI Permeability

Decreased blood flow and ROS exposure can open tight junctions by damaging cell

membranes and promoting necrosis. As a result, GI permeability increases and a cascade

of events occurs, including cytokine release, endotoxin leakage, and monocyte activation.28

Ability for PGs to protect GI mucosal integrity inherently assists in maintaining GI

permeability. However, rather than preventing increased permeability PGs appear to

maintain permeability after damage ensues. Some studies suggest PGs stimulate growth

125

factors that promote epithelial cell repair.155 Others state permeability is maintained by

contracting small intestine villi. Following 15 min of PGE2 exposure, villi height and crypt depth significantly decreased as result of villi smooth muscle contraction (P < .05).

However, negligible effects occurred after 45 min of exposure.156 More recent studies

allude to PG restoring barrier function through closing down tight junctions. An in vitro

study used porcine ileum to examine GI permeability following induced epithelial damage.

Both PGE2 and PGI2 contributed to barrier recovery by collapsing tight jucntions and

tightening lateral membranes of crypt and villi to contiguous cells and basal membranes.157

Another interesting and important finding from this study is the comparison of endogenous

and exogenous PG. Exogenous PGs significantly decreased permeability earlier compared

to endogenous (60 min versus 120 min, P < .01). However, by 180 min following injury

there was no significant difference,157 suggesting both methods are effective at restoring

GI barrier function. In a similar study design, PGI2 and PGE2 acted synergistically to close

tight junctions by increasing intracellular cAMP and calcium signals at the cytoskeleton.158

Vascular Tone and Renal Function

Prostanoids play little role in vascular maintenance in normal healthy individuals.

On the other hand, they are critical in maintaining homeostasis during altered cardiovascular states (eg, hypotension and heart failure) or altered fluid regulation (eg, renal disease, hypovolemia, and sodium imbalance). Prostanoids predominately responsible for vascular maintenance include PGI2, TXA2, and PGE2.

Balance between COX-2 driven PGI2 and PGE2 production plays a significant role

in cardiovascular diseases. Ischemia-reperfusion injury during a cardiac event increases

159 PGI2 and PGE2 expression, which act in a protective manner to promote vasodilation

126

and inhibit platelet aggregation and vascular proliferation.117 Up-regulation of COX-2

following endothelial cell damage stimulates PGI2 in attempt to inhibit artherogenesis. In

160 contrast, PGE2 stimulates atherogenesis due to its strong pro-inflammatory effects. The

strongest opposition to PGI2 is TXA2, which is predominately driven by COX-1 and

promotes platelet aggregation, vasoconstriction, and vascular proliferation.117

The complex relationship between PGE2 and PGI2 on kidney function can be

summarized by three interrelated actions: 1) increased renal blood flow and natriuresis, 2)

vasopressin inhibition, and 3) renin-angiotensin-aldosterone system stimulation. Produced by medullary interstitial cells, papillary collecting tubules, and glomeruli,161,162 renal PGs

respond to hypertension, hypertonicity, and ischemia by stimulating vasodilation and

118,163 increasing renal blood flow. Increased PGE2 production has also been shown to

decrease renal tubular pressure by inhibiting sodium reabsorption through altered cation

164 transport. Prostaglandins, particularly PGE2, inhibit vasopressin by altering cAMP

165 120 activity and through negative feedback. Thirdly, both PGE2 and PGI2 can stimulate

renin and angiotensin II release to cause vasoconstriction and increase BP.118

Inflammation and Pain

Pain sensation begins with peripheral nociceptors in damaged tissue transmitting external stimuli (eg, extreme heat, toxins, or mechanical stress) into electrical energy to be carried along afferent nerves to the spinal cord. Damaged tissues also release

neurotransmitters, particularly substance P or glutamate. Concurrently, IL-1β, TNF-α,

PGE2, PGI2, bradykinin, and numerous other chemical mediators can be released and

potentiate inflammatory pain.39

127

Increased COX-2 expression during injury stimulates PG production,117

116,166 preferentially producing PGE2 and PGI2. These two PGs are responsible for fever,

swelling, redness, and pain – the cardinal signs of inflammation.117,122 Prostaglandins do

not directly cause pain but contribute to local and disperse inflammatory pain by increasing

afferent nerve ending sensitivity to bradykinin, stimulating calcium and sodium channels,

and inhibiting potassium channels. In the spinal cord, PGs stimulate glutamate and

substance P release and activate dorsal horn neurons.39 Coupling mediators (ie, PGs with

bradykinin or bradykinin with substance P) results in an additive effect on vasodilation,

increased vascular permeability, and pain.167,168

Prostanoids regulate inflammation and pain processes in a number of other ways.

Both PGE2 and PGI2 inhibit T cell signaling and differentiation, shifting differentiation

76,122 from Th1 to Th2. Anti-inflammatory PGE2 functions include decreased lymphocyte

proliferation117 and bradykinin stimulation, which is known to protect neurons and inhibit

169 cytokine release. In addition to its potent changes in vascular tone, TXA2 stimulates

lymphocyte activation and proliferation.117 Lastly, cyclopentenone PGs can induce apoptosis by increasing HSP 70 transcription170 and suppress gene expression of

171 macrophage induced TNF-α, COX-2, and PGE2. Each prostanoid plays a unique role in

the body, but in general their response to disease or injury is a vital attempt to maintain

homeostasis. While inhibiting prostanoids may decrease some of the perceived negative

effects (eg, fever, pain, inflammation), inhibition also diminishes any positive, protective

effects.

128

Exercise Induced GI Distress and Inflammation

Intense physical exercise induces a number of GI and acute inflammatory

responses. Some underlying factors that potentiate these responses during exercise are

nutrition, medical history, medication, hormones, dehydration, and Tc. Etiologies for GI

dysfunction and inflammation are complex, and largely influenced by type, duration, and

intensity of exercise.

Type of Exercise

GI Distress

Specific GI symptoms vary across studies, but long distance endurance exercise is

typically associated with more GI distress than anaerobic, short distance events. Among 30

Ironman triathletes (29 males, 1 female, mean age = 33.0 + 6.0 yrs) completing a GI

symptom questionnaire post-event, 93% reported experiencing some type of distress and 2

participants were unable to complete the race due to severe GI distress. Most common

complaints were flatulence (38%, n = 11), belching (35%, n = 10), general stomach

problems (31%, n = 9), and bloating (24%, n = 7).74 In a sample of 125 runners (68 males,

57 females, mean age = 37 + 9 yrs) completing the Marine Corps Marathon, there was a

significant increase from hemoccult-negative stool pre-race to hemoccult-positive stool post-race (n = 29, P < .001).46 Other reported symptoms included abdominal cramps (n =

21), diarrhea (n = 8), and vomiting (n = 1) during or post-race.46 Similar results were found

in 15 ultra-distance runners. Sixty percent (n = 9) reported GI distress, predominately

nausea (89%), abdominal cramps (44%), diarrhea (44%), and vomiting (22%).49

Several studies have compared GI symptom occurrence, duration, and intensity between running and cycling, finding running causes more symptoms than cycling. During

129

a 180min cycle and running protocol in moderate temperature, 95% of participants (n =

21/22) reported GI symptoms during exercise. Running resulted in significantly more symptoms than cycling (P < .05), specifically belching, heartburn, cramping, and side aches.172 A similar run-cycle-run protocol in 7 well trained male athletes resulted in significantly more reflux episodes during running than cycling.43 Gastrointestinal motility was significantly higher with cycling compared to rest (6.1 versus 4.7, P = .025) and carbohydrate beverage transit time progressively increased across rest, cycling, and running (138min, 193min, 240min, respectively, P = .03).173

Inflammation

Inflammatory responses during exercise include increased circulating pro- inflammatory cytokines (IL-1β, IL-6, and TNF-α), PGE 2, HSP70, and increased neutrophil and lymphocyte (T4 cell, T8 cell, and NKC) chemotaxis.174-176 Plasma IL-6 levels rise relatively quickly during intense exercise, peaking immediately after. In contrast, TNF-α responses are slower and peak hours post-exercise.174 In 9 sedentary males completing 45 minutes of downhill running IL-1β, PGE2, and neutrophil chemotaxis significantly increased after exercise.177,178 No increase in IL-6 and TNF-α was found,177 which may be attributed to a short duration and lower exercise intensity. In 16 male marathon runners

(mean age = 30.5 + 1.9 yrs, V̇ O2max = 43.6 + 2.1 ml/kg/min) IL-6 significantly increased baseline to post-race (1.5 + 0.7 pg/ml to 94.4 + 12.6 pg/ml, P < .0001). Pre- to post-race

IL-1β (0.61 + 0.24 pg/ml to 0.92 + 0.26 pg/ml, P < .005) and TNF-α (0.96 + 0.09 pg/ml to

2.43 + 0.37 pg/ml, P < .0001) also significantly increased.176 Nine male Ironman triathletes competing in moderate temperatures (mean = 23.3 + 1.9°C) had significantly increased plasma HSP70 and IL-6 post-race (P < .001).175

130

Potential Etiologies

Research is inconclusive regarding exact etiology of exercise induced GI and

inflammatory responses. In part this is due to varying methodologies among controlled

laboratory studies and the limitations of field studies. Potential etiologies include damage

caused by mechanical vibrations, ischemia-reperfusion injury, and increased GI

permeability with LPS translocation, but a combination of these factors is likely.

Gastrointestinal dysfunction during physical activity may be caused by mechanical

vibrations and increased abdominal pressure.179 There is limited research in this area and

the existing literature has several limitations. Using accelerometers on 6 well trained

participants (3 males, 3 females) during separate cycling and running bouts resulted in

significantly greater output during running (mean counts/min = 859.5+ 130.1 versus 425.8

+ 149.5, P < .0001).38 Differences within activity affect vibrations as well. Running shoe

components and surface type can increase or decrease vibrations transferred to the GI.

Cycling position, such as standing, sitting, and leaning can also affect vibrations.38

A profound shunting of blood during intense exercise results in local and systemic

inflammatory responses. Ischemia occurs when splanchnic blood flow decreases more than

50%23 and is often considered the primary cause of GI damage. Epithelial cells are

particularly vulnerable. In hypoxic states they produce ROS and cytokines, attract

neutrophils, and experience altered basal membrane attachment.41 Responses to GI ischemia-reperfusion impairs tight junctions, ultimately increasing permeability and LPS translocation.180

Intense exercise during field and controlled laboratory studies increases GI

peremability.61,181,182 Percent recovery of lactulose/rhamnose was significantly higher

131

182 during a 60 min run at 80% V̇ O2max than lower intensities and rest (P < .05). With

hydration controlled in a thermoneutral environment, 10 participants (9 males, 1 female)

completed a rest, cycling, and running protocol. Small intestine permeability was

significantly higher during running (mean lactulose/rhamnose ratio = 0.04 + 0.02; P < .01)

compared to cycling (0.03 + 0.02) and rest (0.02 + 0.02).173

Similar results are indicated with plasma LPS measures. During cycling to

exhaustion in 10 males plasma LPS significantly increased pre- to post-exercise (0.14 to

0.24 Eu/ml, P < .01).72 Higher incidence of vomiting, diarrhea, and nausea were reported

in marathon runners with elevated LPS measures (0.33 + 0.04 ng/ml) compared to those

with low LPS (0.08 + 0.01 ng/ml, P < .001).61 In attempt to identify if GI distress is

associated with ischemia driven endotoxemia and inflammatory cytokines, blood measures

were taken 24 hours pre-Ironman, immediately post-race, and 1, 2, and 15-20 hrs post-race.

A significant increase from pre- to 1 hour post was found for mean LPS and IL-6 concentration (0 pg/ml to 4 pg/ml, and 0 pg/ml to 40 pg/ml, respectively, P < .05). No GI symptoms were correlated with LPS concentration. However, some symptoms were significantly, albeit weakly, correlated to IL-6 (vomiting r2 = 0.268 and diarrhea r2 = 0.504,

P < .05).74

Heat Stress Induced GI Distress and Inflammation

The greatest GI response to non-exertional thermal stress is impaired barrier

function and subsequent LPS translocation. In 12 primates subjected to passive

hyperthermia (41 + 0.3°C, 100% relative humidity), blood LPS measures significantly

increased from 0.06 + 0.01ng/ml to 0.32 + 0.03ng/ml (P < .01).183 In non-EHS patients

(mean Tc = 42.1 + 0.2°C), significant decreases pre- to post-cooling were found for mean

132

plasma TNF-α (247.4 + 41.2 pg/ml to 133 + 27.9 pg/ml, P < .05) and LPS (12.26 + 1.86

ng/ml to 6.12 + 1.18 ng/ml, P < .05).84 Raising Caco-2 human intestinal epithelial cell

temperature from 37°C to 41°C cultures resulted in a strong, linear, inverse relationship (r

= 0.96) between temperature and transepithelial resistance,184 a measure of tight junction

integrity. These results, along with HSP research suggest thermal stress induced

permeability changes occur at tight junctions. Following 2hrs of heat exposure (Caco-2 cell

temperature = 41°C) HSP70 expression peaked 4-8hrs post and tight junction protein

expression decreased. When HSP was inhibited paracellular permeability was significantly

greater (P < .001) than no HSP inhibition.184

A number of other inflammatory mediators are released during passive

hyperthermia. In particular, elevated IL-6 (220 + 44 pg/ml) was identified in 18 non-EHS

patients with a mean Tc = 41.04 + 0.2°C.185 Another study showed pre-cooling IL-6

concentration and severity of passive heat stroke was significantly correlated (r = 0.516, P

< .03).186 In patients experiencing non-EHS, TNF-α significantly decreased pre- to post- cooling (247.4 + 41.2 pg/ml to 133 + 27.9 pg/ml, P < .05) and remained above control

values 31.4 + 8.4 pg/ml. Working synergistically, TNF-α and IL-6 may exacerbate

hyperthermia.187 If combined with exercise, GI and inflammatory responses during thermal

stress may lead to serious, potentially lethal conditions of endoxtemia, SIR, or EHS.

EXERTIONAL HEAT STROKE

Individuals may experience a number of symptoms when heat dissipation

mechanisms are compromised during intense exercise in extreme thermal environments.

Initially, minor symptoms such as dizziness, lightheadedness, or fainting may occur, which

are typical of heat syncope. Heat syncope occurs due to decreased brain blood flow as a

133

result of peripheral vasodilation, postural blood pooling, diminished venous return, cardiac

output reduction, and/or cardiac ischemia.9,188 A more serious EHI, heat exhaustion can occur as the cardiovascular fails and plasma volume decreases. Heat exhaustion symptoms include dizziness and lightheadedness, as well as fatigue, headache, normal-to-elevated Tc

(< 40°C), nausea, vomiting, intestinal cramps, tachycardia, and/or hyperventilation.

Complete thermoregulatory and cardiovascular system failure results in EHS.4 Excessive

metabolic heat production dangerously increases Tc, and combined with decreased brain

blood flow, the central nervous system is impaired and organ damage ensues.

Uniqueness and Individuality of Exertional Heat Stroke Cases

Exertional heat stroke is identified by 2 key characteristics: 1) a Tc > 40°C and 2)

alterations in central nervous system function.9 It is important to note that 40°C is not a

“golden number”. It remains unclear why some individuals can participate in intense

activity with Tc above 40°C without any EHS symptoms and other individuals experience

EHS below 40°C. Some variability can be explained by physical conditioning,

acclimatization, body mass, hydration, equipment or clothing, and sleep deprivation. These

factors are well understood in regard to physiological strain and disseminating metabolic

heat. Untrained, unacclimatized individuals with larger body-surfaces have decreased thermal tolerance.189 Dehydration impairs cardiovascular function, increasing Tc and

perceptual thermal strain measures.190 Equipment and clothing creates a microenvironment

that limits heat dissipation189

Previous illness and medication use are known risk factors for EHS, but less

understood because controlled laboratory studies in human subjects are not available.

Insight into the impact these factors play in EHS primarily comes from case and

134

observational studies. A retrospective assessment of 38 EHS deaths among American

college and high school football players identified 1 player who experienced a GI virus the day before and 1 who had a history of taking ephedra.191 A 19yr old male participating in

intense exercise in the heat experienced multiple heat-related incidences. The first episode

was precipitated by gastroenteritis.192 Clinical observations of 36 male EHS patients found

1 experienced a fever the week prior, 6 presented with GI distress within 3 days of the

event, and 1 had EHS 3 days earlier. In addition to the 6 with previous GI distress, 16

patients experienced GI distress during the EHS.193 In one of the most publicized EHS deaths in American football, the athlete experienced GI distress (“upset stomach” and vomiting) the day prior to and day of his death.194

Etiology: Models of Exertional Heat Stroke

The hypothalamus is considered the body’s thermostat. Individual baseline Tc may

vary slightly, but the hypothalamus attempts to maintain Tc at about 37 + 1°C. Unlike a

thermostat, the hypothalamus cannot turn off heat and can only initiate responses designed

to mitigate rising Tc. Hypothalamic driven mechanisms for heat loss include stimulating

evaporative heat loss and cutaneous blood vessel vasodilation.1 Inability for the

hypothalamus and thermoregulatory responses to rid heat gain can dangerously increase

Tc.

Heat transfer occurs by movement from warmer to cooler objects/environments.

Sun exposure creates radiation heat gain. Radiation heat loss occurs when the body is

warmer than surrounding air.19 Conduction involves movement of heat energy with two

objects in contact with one another. For example, warm skin in contact with cold water

allows heat to dissipate from the body to the water. Similarly, convection is heat exchange

135

between moving air/fluids, and is impacted by movement speed. Using the previous

conduction example, when no movement occurs the water in contact with the skin will

become warm and the skin will cease to dissipate heat. Moving the water across the skin

will allow for cooler water to pass by, promoting additional heat dissipation. Less heat is

lost with slower movement. Evaporation occurs through respiration and sweating and is

the major defense against heat gain.1

Early Theory of Exertional Heat Stroke

Exertional heat stroke is typically thought to occur due to metabolic heat gain from

working muscles and inability to dissipate heat to the surrounding environment.1 During

fever, infection, or passive hyperthermia elevated Tc increases metabolic rate. However,

during intense physical exercise metabolic heat from chemical and mechanical actions

raises Tc.19 In effort to promote heat loss the hypothalamus induces skin capillary

vasodilation to deliver blood throughout the circulatory.195 This also increases the demand to dissipate heat through evaporation, conduction, and convection.4

High environmental temperature and humidity are major limiting factors for

effective heat loss. The greatest EHS risk occurs when wet bulb globe temperatures exceed

28°C,189 but has been reported in cooler environments (6 – 9.5°C).196 Through convection

and conduction, the body will gain heat when environmental temperature is higher than

body temperature. This requires a high demand in evaporative heat loss, seen with elevated

sweat rates proportionate to elevated temperatures. In the presence of high humidity evaporative sweat loss is limited.1 The combination of metabolic heat production during intense exercise and inability to dissipate heat exceeds the body’s thermoregulatory response. As a result, Tc increases and an individual is susceptible to experiencing an EHS.

136

Dual Model of Exertional Heat Stroke

More recently, the prevalence of previous illness and GI distress in EHS cases has

led some to speculate that the GI tract and immune system are key underlying mechanisms.

These general similarities have led researchers to examine a closely intertwined dual model

for developing EHS. Both models are largely influenced by GI and immune responses due

to prolonged exposure to intense exercise or hyperthermia during exercise.5

First Model of Exertional Heat Stroke: Hyperthermia during Exercise

The first EHS model is due to exercise and thermal stress. An increase in heat

storage causes GI distress and increased GI permeability.5 Exacerbated by GI ischemia and

inflammatory cytokines, increased permeability allows LPS to escape into blood

circulation.5,197 Exercise can continue as long as circulating LPS is cleaned up through previously mentioned immune and liver responses. However, once the liver is overwhelmed, endotoxemia occurs and additional pyrogenic cytokines are released.5 At

higher temperatures, such as with intense exercise, both pro- and anti-inflammatory

cytokines are released. A somewhat futile immune system results, with the body producing

anti-inflammatory cytokines to counteract endotoxin and pyrogenic cytokines.

Unfortuantely, the pro-inflammatory cytokines (IL-6 and TNF-α) are necessary to neutralize endotoxemia.187 Cytokines also promote necrosis, vasodilation, acidosis,

decreased plasma volume, increased vascular permeability, and fever.185,193,198 The end result is SIR, multi-organ failure, and/or EHS.5

Second Model of Exertional Heat Stroke: Prolonged Exposure to Intense Exercise

The second model states regardless of environmental conditions, prolonged intense

exercise is immunosuppressive. Down-regulation of Th1, up-regulation of Th2, and

137

decreased NKCs contribute to blunted immune responses. In the event a pathogen is

introduced, a sub-clinical infection develops5 where the individual may or may not

experience symptoms.192 At this point, the individual attempts another bout of intense

exercise, potentially in a thermal or thermoneutral environment. The compromised immune

system and increased cytokines limits the liver’s ability to neutralize circulating LPS. Like

the first model, if uncontrolled, endotoxemia, SIR, or EHS may occur.5

An example of this model may be observed in individuals participating in

successive intense exercise bouts (eg, pre-season football practices). The individual’s

immune system is disturbed after the first intense exercise bout. On the following day, the

individual attempts another bout of intense physical activity. However, GI damage and a

compromised immune system decreases his/her thermal and work capacity. Unable to

mitigate LPS, and with elevated pyrogenic cytokines, endotoxemia develops, Tc rises, and

the person experiences an EHS.

It is possible to develop EHS without experiencing endotoxemia. Animal studies

have shown blocking LPS protects against heat stroke until a Tc > 43.8°C.199,200 Extremely high temperatures denature proteins and cause cell necrosis.5 Since EHS does not always

occur in hot environmental temperatures and some individuals can exercise at 40°C with

no EHS symptoms, the dual model provides insight into underlying GI and inflammatory

factors that contribute to EHS. Furthermore, risk factors (eg, previous illness, medication,

sleep disturbances, and nutrition) that alter GI integrity and immune responses play roles

in the dual model.

138

NON-STEROIDAL ANTI-INFLAMMATORY DRUGS (NSAIDs)

Non-steroidal anti-inflammatory drugs (NSAIDs) include a large variety of

prescription and non-prescription medications including acetylsalicylic acid (aspirin),

ibuprofen (eg, Advil, Motrin), indomethacin (eg, Indocin), naproxen (eg, Aleve,

Naprosyn), and numerous others. Prevalence of NSAID use among the general public is

high, spending billions on over-the-counter pain medication.201 Consumer reports and advertisers suggest ibuprofen and naproxen are safe, affordable, and effective pain medications,202 which is supported by 2013 revenue reports including both Advil

(ibuprofen) and Aleve (naproxen sodium) in the top 10 over-the-counter brands.203

Prevalence of medication use among elite athletes has been well documented due

to World Anti-Doping Agency and International Olympic Committee drug policies.

Medication, particularly NSAIDs, is a concern across all age groups, sport type, and

competition level. Physically active persons use NSAIDs prophylactically or following

onset of inflammation, pain, and/or fever.204-208 Despite an injury, athletes may choose to

use NSAIDs in order to continue participation or because they perceive it will help their

performance.208 Among 3,887 elite international track and field athletes completing a 7 day

recall of medication use, 3,237 reported taking medications, with COX-1 inhibitors the

most common (27.3%, n = 782).206 In a similar study, of 912 elite athletes across 34 sports completing 3 day recall doping control forms, 24.7% (n = 225) reported NSAID use.207 Of particular interest, 22% (n = 50) reported taking multiple NSAIDs at one time.207

Combining NSAIDs is not advised, particularly due to potential adverse drug reactions209

and increased risk of GI damage.210

139

Similar prevalence of NSAID use has been reported in recreational athletes. Of 333

ultra-distance triathletes, 100 (30%) reported taking NSAIDs within the 24 hours prior to

the race.211 Less research has been conducted among youth and team sport athletes.

However, of the available studies, prevalence of use is similar or even higher than in adults.

Of 604 high school football players reporting medication use over the past 3 months, 75%

(n = 452) reported using NSAIDs related to their sport participation.208 Two hundred thirty-

two independently administered NSAIDs (i.e. no adult supervision) and only 51 perceived

any disadvantages to taking NSAIDs.208 Although this was a limited population, studies among the general population suggest adolescents often self-administer NSAIDs212,213 and are unaware of potentially toxic side effects.214,215

NSAIDs are perceived as performance enhancing because they mitigate pain and

inflammation. Differences between medication type, dosage, and exercise mode in existing

literature make it difficult to conclusively state whether NSAIDs do or do not enhance

performance. However, aspirin does not alleviate exercise induced muscle pain,216 and may

degrade endurance exercise performance by increasing lactate production and decreasing

time to fatigue.217 Results are similar in upper and lower extremity resistance exercise,

finding aspirin increased rate of perceived exertion (RPE) and perceived leg pain versus a

placebo.218 Further supporting the lack of performance enhancing effects, compared to

placebo, aspirin had little effect on respiratory variables (respiratory rate, oxygen

consumption, carbon dioxide, etc.), HR,217 anaerobic threshold onset, and work load.219

Mechanisms of Action

All NSAIDs work to reduce inflammation and pain, but their mechanisms of action

varies depending on chemical structure. Consequently, their effectiveness as anti-

140

inflammatory and anti-pain medications, along with their side effects, also varies greatly.

The plant based salicylic acid (acetylsalicylic acid is a derivative) has been used for

thousands of years to reduce inflammation and pain, long before its mechanism of action

was understood. It was not until the 1970s researchers identified aspirin and all other

NSAIDs’ mechanism of action – inactivation of COX.220,221 By inhibiting COX, and therefore PG synthesis, NSAIDs decrease inflammatory responses and inflammatory induced pain. An NSAID’s mechanism of action can be further defined as either “non- selective” or “selective”, where non-selective inhibit both COX-1 and COX-2 and selective target COX-2 only.222

The most common way to determine a drug’s selectivity is to measure the half

maximal inhibitory concentration (IC50). This value represents the drug concentration

needed to inhibit a substance or function by 50%.223 A ratio of COX-1 to COX-2 inhibition is determined by dividing the IC50 value for COX-2 in to COX-1. It is important to note

that drug selectivity is a continuous variable; there is no absolute cut-off value to classify

223,224 selective versus non-selective. Utilizing IC50 ratios may allow for more standardized

comparison and classification220 within a study in regards to effectiveness and side effects.

A drug is generally considered to be more COX-2 selective if it has a lower IC50 ratio.

Unfortunately, within and between studies there is variability in using whole blood or cell

cultures, assays, incubation time, and substrate concentration.225,226 Direct comparison across studies is not recommended. Instead, ranking NSAID selectivity relative to IC50

ratios (eg, most COX-1 selective to least) is commonly seen. While this allows for some

generalization across studies, when reviewing rankings one would also see large variability

in IC50 ratios for a given drug.

141

Non-Selective COX Inhibitors

Majority of NSAIDs, particularly those available over-the-counter, are non- selective COX inhibitors. Due to different chemical compositions, inhibition of COX-1 and COX-2 is not equal and each NSAID has its own selectivity toward one COX variant or the other. A “non-selective” NSAID may be more COX-1 selective, while still inhibiting

COX-2 to a certain percent. Choosing one NSAID over another is often dependent on desired therapeutic effect and patient response to a given drug.

Acetylsalicylic Acid (Aspirin)

Acetylsalicylic acid is the most widely used and researched NSAID, partly because it is oldest and inexpensive. Its COX inhibition is heavily time dependent, with rapid liver enzymatic activity resulting in a relatively short 2-3 hour half-life.227 Acetylsalicylic acid

is indicated for mild to moderate pain, fever, and reducing risk of cardiac events.227,228 Its

229 ability to irreversibly inhibit platelet driven TXA2 production by almost 95% with low-

doses (30 mg per day) make it highly effective at preventing blood clots and reducing risk

of heart attacks and strokes.229,230 Due to greater selectively toward COX-1 than COX-

2,18,222 much higher doses (320 mg per day)231 are recommended to achieve anti-

inflammatory effects.229 Research using recommended over-the-counter doses has been unable to identify substantial anti-inflammatory effects. Following endotoxin injection, oral ingestion of 1000 mg resulted in no change in circulating TNF-α and IL-6 versus a placebo in resting patients.232

Ibuprofen

Ibuprofen is another commonly used NSAID and the first non-aspirin over-the-

counter NSAID.228 Similar to acetylsalicylic acid, ibuprofen is indicated for mild to

142

moderate pain, fever, and inflammatory relief and has a relatively short half-life of 2-4

hours.227 Ibuprofen is considered equipotent against both COX derivatives.233 Extensive

research has been conducted on inflammatory effects during various exercise bouts234,235

or following endotoxin injection.236-238 Results are conflicting, with some studies showing

ibuprofen has significant235-238 or no effect on inflammatory markers.234,235

Contradictions in exercise studies can be partially explained by interpersonal differences of participants (eg, history of medication use and illness, age, acclimatization,

and conditioning) and varying methodology (eg, exercise duration, intensity, environment,

and medication dosage). A one-time 400 mg dose prior to 1500 m run in unknown

environmental conditions showed no significant difference in TNF-α, IL-6, or IL-1β pre-, immediately post-, or 24 hours post.234 On the other hand, 29 runners completing a 160km

ultramarathon in moderate temperatures (10°C-25°C, 56% relative humidity) were given

600 mg of ibuprofen the afternoon prior to the race and another 1,200mg on race day (one

200 mg tablet every 4 hours). Compared to no medication, most inflammatory markers,

including IL-6, significantly increased pre- to post-race; however, there was no significant

change in TNF-α.235

Results from endotoxin injection studies at rest are slightly more consistent. As

expected, ibuprofen blunts Tc rise following endotoxin challenge, but has been shown to

increase inflammatory markers. Compared to control, two 800mg ibuprofen doses pre-

endotoxin injection significantly increased IL-6 (27 + 12 ng/ml vs. 113 + 66 ng/ml, P =

.05, respectively) and TNF-α (369 + 44 pg/ml vs 627 + 136 pg/ml, P = .003,

respectively).237 Similar results were found after subjects were given three 800mg doses

(1.5 hours pre-, at the time of, and 3hrs after endotoxin challenge).238

143

Indomethacin

Indomethacin is typically used to control moderate pain in patients who do not

respond to other, less toxic NSAIDs. Absorption is variable depending on administration

route, and average half-life is 4-6 hours.227 Like other non-aspirin NSAIDs, it is less

effective at reducing cardiovascular events.239 However, it is a potent inhibitor of COX-

1240 making it useful as an anti-inflammatory agent. In 10 recreational athletes, indomethacin significantly blunted post-exercise NKC suppression, IL-6 expression, and

TNF-α expression (P < .05).241,242

Naproxen

Naproxen is a relatively newer drug with less selectivity toward COX-1 than aspirin

or indomethacin.243,222 Naproxen is available in either free acid or sodium form.244

Naproxen sodium is commonly seen as Aleve® or naprosyn. The two forms are

bioequivalent on all levels except rate of absorption. Gastric juice rapidly dissolves

naproxen sodium, resulting in high plasma concentrations within 1 hr compared to 2 hrs

with naproxen. The earlier onset of symptom relief with naproxen sodium is beneficial for

acute pain.244 Over-the-counter doses (200 mg or 400 mg) are effective for mild to

moderate pain, fever, and inflammation. Compared to other NSAIDs, naproxen has a long

half-life of 10 to 20 hours.227 While a patient may need to take aspirin or ibuprofen every

4 hours to maintain therapeutic effects, patients using naproxen are instructed to take 1

dose every 8 hours and to not exceed 600 mg per day.227

In regards to therapeutic effects, 500 mg of naproxen sodium twice a day has been

shown to inhibit platelet COX-1 activity close to 95%. However, platelet inhibition is

reversible and inconsistencies between subjects make it a less than ideal choice as a cardio-

144

245,246 247 protective medication. Naproxen significantly inhibits PGE2 and PGI2, making it

useful in a variety of inflammatory and pain diseases.244

Selective COX-2 Inhibitors

Three popular selective COX-2 inhibitors, also known as coxibs, are rofecoxib

(Vioxx), valdecoxib (Bextra), and celecoxib (Celebrex). Meloxicam (Mobic) was not

originally designed to be a COX-2 inhibitor, but research has shown it to be very COX-2

specific.248 With little inhibition of COX-1, these NSAIDs are more effective as anti- inflammatories while reducing toxic GI side effects.222

Adverse Effects of NSAIDs

Gastrointestinal Effects

Risk and prevalence of adverse GI effects from NSAIDs is well established among published cases, multicenter case-control studies, and randomized control trials.17,69,243,249-

251 Time from initial NSAID use to onset of GI distress varies from days to several years252

and risk increases with higher doses, longer use, longer half-life, slow-release formulation, greater COX-1 selectivity, and previous adverse events.243 In general, NSAIDs,

particularly non-selective, cause adverse effects by inhibiting COX-1 and, therefore, GI

protective PG effects – increased mucus and bicarbonate secretions, increased blood flow, decreased acid secretion, TJ maintenance, etc. Selective COX-2 inhibitors were developed to create a NSAID that reduced GI effects by preserving COX-1. However, research has shown both COX derivatives play a role in GI protection253 and NSAIDs may directly

induce GI distress through non-PG mechanisms.16

145

Gastrointestinal Damage

Specific GI damage includes colitis and ulcerative colitis; peptic ulcers; small and large intestine perforation, hemorrhaging and abscesses; and small intestine strictures.252,254

The relationship between the spectrum of COX inhibition and relative risk (RR) for GI damage has been illustrated in a meta-analysis comparing common NSAIDs and doses.

Lowest risk was found for the more COX-2 selective celecoxib (RR = 1.42, 95% CI 0.85

– 2.37) and ibuprofen (RR = 2.69, 95% CI 2.17 – 3.33). The highest risk was found in the greater COX-1 specific ketorolac (Toradol; RR = 14.54, 95% CI 5.87 – 36.04).

Interestingly, compared to no use, NSAIDs inhibiting both COX derivatives by greater than 80% resulted in a higher GI damage risk; this included naproxen (RR = 5.63, 95% CI

3.83 – 8.28) and indomethacin (RR = 5.40, 95% CI 4.16 – 7.00).243 In a comparison of GI toxicity among 8,076 rheumatoid arthritis patients, naproxen use elicited significantly more upper GI damage (ulcers, obstruction, perforation, and bleeding) versus rofecoxib (121 vs

56 events, P < .001).249 Thirty-six healthy participants (24 males, 12 females, mean age =

48 yrs) with no previous NSAID intolerance were used to compare 500 mg naproxen versus

100 mg nimesulide (a selective COX-2 inhibitor) administered twice a day for two weeks.

Naproxen significantly inhibited PGE2 and PGI2 (P < .001) and caused stomach damage

(hemorrhage, lesions, and ulcers) in 24 participants compared to 2 in the nimesulide group.250

There is a lack of research on lower GI damage and results are less consistent regarding selectivity. In 34,701 rheumatoid and osteoarthritis patients receiving either diclofenac (non-selective) or etoricoxib, bleeding, perforation, obstruction, diverticulitis, and ulcers were reported, but there was no significant difference between treatment

146

groups.255 One the other hand, relative risk for developing serious lower GI damage was significantly greater with naproxen use compared to rofecoxib (0.46, P = .032).256 A 43 year old female patient with low back pain regularly taking diclofenac (Voltaren) and intermittently using ibuprofen and indomethacin reported to a hospital with hematochezia.

Examination and biopsy revealed ulcers along the ascending colon, cecum, and ileum. Two months after cessation of NSAIDs a follow-up colonoscopy revealed normal mucosa.257

A concurrent inflammatory response may exacerbate GI damage. Intestinal edema,

lymphocyte and neutrophil accumulation257 and increased cytokines is associated with

gastric lesions. Oral administration of indomethacin and aspirin in rats significantly

increased TNF-α and gastric lesion scores compared to control (P < .001). Pre-treatment

of a TNF-α inhibitor significantly decreased indomethacin induced gastric lesions (P <

.05).258

Gastrointestinal Permeability

Several randomized, blinded control trials have shown different NSAIDs increase

GI permeability within hours and after single doses. In 14 healthy participants, GI

permeability, measured using Cr-EDTA, significantly and linearly increased with potency

after 2 single doses of aspirin (2.3 + 0.3%, P < .05), ibuprofen (2.9 + 1.2%, P < .001), or

indomethacin (4.7 + 1.3, P < .001) compared to baseline (1.9 + 0.5%).259 Nineteen healthy

females were used to determine differences in permeability following naproxen,

meloxicam, indomethacin, or celecoxib. Compared to no NSAID, only naproxen resulted

in significantly higher gastroduodenal permeability (median sucrose excretion = 78.4 mg

[51.4 – 105.4 95% CI] versus 107 mg [82.9 – 138.5 95% CI], P < .05). In regards to small

intestine permeability, naproxen significantly increased the lactulose/mannitol ratio (0.032

147

versus 0.022 [baseline], P < .05) while celecoxib had no significant effect.251 In 8

participants (6 males, 2 females; mean age = 22.5 + 2.5 yrs; mean V̇ O2max = 61.8 + 5.7

ml/kg/min) running 60 min in a thermoneutral environment, aspirin and ibuprofen

significantly increased gastroduodenal permeability compared to placebo (% sucrose

secretion = 0.37, 0.22, and 0.09, respectively, P < .05). Only aspirin significantly increased

small intestine permeability versus placebo (lactulose/rhamnose ratio = 0.09 versus 0.065,

P < .05).14 An attempt to determine aspirin dosage needed to induce GI permeability found

3 doses of 325 mg significantly increased gastroduodenal permeability (P < .05).260

Overall, research supports acute, over-the-counter NSAID doses alter GI permeability.

Some question has been raised against the generally accepted theory that NSAIDs

induce permeability changes due to PG inhibition. Using 7 participants to determine if

increased GI permeability was driven by COX inhibition, co-administration of PGE2 and

indomethacin did not result in significant differences in excreted Cr-EDTA compared to indomethacin alone (6.6 + 4.4% and 4.6 + 1.5%, respectively).259 Similar results were

found among 40 healthy participants administered indomethacin, indomethacin and

exogenous PGE2, or indomethacin and metronidazole (an antibiotic). Co-administration of

PGE2 failed to prevent increased GI permeability. On the other hand, metronidazole significantly inhibited GI permeability compared to indomethacin alone (P < .05).261

Inability of PG to emolliate NSAID induced GI damage does not rule this mechanism out,

particularly considering the limitations to exogenous PG use and other studies finding PG co-administration reduces GI permeability.262 However, it does allude to non-PG mediated

mechanisms.

148

The GI mucosa is hydrophobic, preventing potentially harmful molecules from

passing through.263 Altering hydrophobicity can increase GI permeability. Compared to saline, aspirin significantly decreased mucosa hydrophobicity among wild-type and COX-

1 deficient mice (P < .05). This was associated with significantly higher gastric lesion

scores (P < .05).16 Further support comes from a group of researchers showing aspirin,

ibuprofen, and naproxen interact with phosphatidylcholines (a group of constituent

phospholipids in cell membranes). This interaction disrupts TJs and creates unstable pores

by altering hydrophobicity, fluidity, and cell structure on mucosa surface and at the lipid

bilayer.264,265

Other suggested mechanisms are less researched and focus around NSAIDs

reducing ATP production. The ability for metronidazole to maintain GI permeability in the

aforementioned study suggests an inflammatory mediated mechanism.261 By reducing

ATP, presumably through glycolysis and tricarboxylic acid cycle inhibition, there is a

disruption of the cytoskeleton and loss of TJ regulation, increase in ROS production, and

protein degradation.15, Results on glucose supplementation maintaining permeability are

conflicting. Co-administration of an indomethacin, glucose, and citrate formula blunted

permeability changes compared to indomethacin.266 In contrast, no difference in permeability occurred with 1300 mg of aspirin and replenishing glucose through a carbohydrate beverage.15

Cardiovascular Effects

As discussed earlier, PGs play an important role in maintaining cardiovascular

function, producing prostanoids to maintain BP and blood volume. Selective COX-2

inhibitors have greater cardiac risks. Like GI effects, degree of selectivity plays a role on

149

occurrence of cardiac events, meaning even non-selective NSAIDs cause adverse cardiac effects.246,267 Aspirin is cardioprotective, preventing blood clots by irreversibly inhibiting

268 TXA2, but aspirin is also linked to increased bleeding time. Both selective and non-

selective NSAIDs have been shown to increase myocardial infarction246 and stroke risk.267

Lastly, inhibiting COX driven vasodilation decreases skin blood flow,269 which limits the

body’s ability to dissipate heat during exercise or thermal strain.

Renal Effects

In addition to altering renal function through cardiovascular effects, NSAIDs

directly impact kidney function by inhibiting renal PGs, which maintain renal blood flow.

Serious side effects include papillary necrosis and interstitial nephritis.118 Less serious side effects are fluid-electrolyte imbalances such as hyponatremia (plasma sodium concentration (P[Na+]) < 135 mmol/L) and renal hypertension. If coupled with volume

depletion, a potential risk during exercise,34 these effects can be exacerbated and lead to

acute renal failure.270 By inhibiting PGs, NSAIDs suppress the renin-angiotensin-

aldosterone system, decrease glomerular filtration rate, increase vasopressin, and increase

sodium retention. As a result, water is retained and vasoconstriction occurs, leading to

edema and hypertension.271,272

Decreased urine production with increased water retention from NSAID use is

considered a contributing factor for hyponatremia. Thirty percent of ultradistance

triathletes (n = 100/333) reported using NSAIDs and had significantly lower post-race

plasma sodium compared to athletes not using NSAIDs (mean = 140.2 mmol/L versus

141.1mmol/L, P < .02). The 6 participants who experienced hyponatremia during the race

all took NSAIDs (χ2 = 14.24, P = .0002).211 Out of 60 hospitalized marathon runners, 15

150

experienced severe hyponatremia. Percent NSAID use was significantly higher compared

to normonatremic runners (28.6% versus 4.6%, P = .010).273 Other observational studies

have not identified a relationship between NSAIDs and hyponatremia.274,275 Controlled

laboratory studies are lacking and observational studies have inherent limitations.

Nevertheless, it is known NSAIDs adversely affect fluid-electrolyte balance. Additional

cardiovascular and thermoregulatory strain limits ability to dissipate heat and increases

EHI risk.

NSAIDs: A Risk Factor for Exertional Heat Stroke?

Due to their anti-pyretic effects, taking NSAIDs during exercise and/or thermal

stress has been speculated to lower Tc, but current literature is limited and inconclusive.

After taking 7,800 mg of sodium salicylate or a placebo, 12 trained males walked for 100

min at 3.8 mph in a hot, dry environment (48.9°C dry bulb, 26.7°C wet bulb) and 16 in a

hot, wet environment (33.3°C dry blub, 30.6°C wet bulb). No significant differences in Tc

were recorded between placebo and sodium salicylate in either environmental condition.

Though not significant, Tc with salicylate began to increase compared to placebo at 40 min

(hot, dry) and 30 min (hot, wet), continuing until the exercise protocol ended.276 A possible explanation for lack of significance between Tc could be the exercise protocol intensity.

Mean HR was not significantly different between control (152 bpm) and NSAID (159 bpm) and below the established cut-off of 180 bpm to cease walking.276 An earlier study administered acetylsalicylic acid in either 1 dose, 48 hours, or 7 days on 10 males (age range = 23 – 26 yrs). Participants completed a short 20 min walk at 3.5 mph and 8.6% grade in a temperate environment (22.2°C, 50-55% RH). Resting, exercise, or recovery Tc was not significantly different across experimental groups and did not rise above 38°C.277

151

Ten male participants taking 6 days of rofecoxib or placebo completed an exercise protocol at 75% V̇ O2max in temperate conditions (28°C, 50% RH). No significant difference was found in Tc between experimental conditions during the first 45 minutes of exercise

(running). Experimental participants exhibited significantly lower Tc during the last 45 minutes of cycling (mean difference = 0.33 + 0.26°C, P < .05) and throughout a 60 min recovery (mean difference = 0.34 + 0.26°C, P < .01). Overall, TNF-α significantly increased pre- to post-exercise (2.96 + 2.07 to 4.17 + 3.25 pg/ml, P = .03), but no difference was found between groups. Change in skin blood flow from rest to cycling was significantly higher in the experimental group versus placebo (P = .01).278 Together, these results suggest rofecoxib blunts Tc rise by increasing skin blood flow. This is in contrast to a recent study using aspirin. In a randomized, double-blind, crossover study (n = 14, 7 males, 7 females, mean age = 55 + 1 yr), the effects of 7 days of low dose (81mg) aspirin on Tc, skin blood flow, thermal sensation, and hydration measures were compared to placebo and PlavixR (a prescription anti-platelet medication). Participants rested 40 min in a thermal environment (30°C dry bulb, 22°C wet bulb, 40% RH) then biked 2 hours at 60%

V̇ O2peak. Following rest, Tc was significantly higher for aspirin compared to placebo (P <

.05) and remained significantly higher throughout exercise (P < .001).269 Rate of temperature rise, thermal strain, HR, and plasma volume were not significantly different between experimental groups, but skin blood flow was significantly decreased with aspirin use (P < .05).269 Attenuating skin blood flow, thus limiting heat dissipation, is elevates Tc.

SUMMARY AND CONCLUSION

Literature supports a significant role of the GI tract and immune system in development of EHS. Together or alone, intense exercise and thermal stress increase GI

152

permeability, LPS translocation, and inflammatory responses. The result is increased Tc

and EHS risk. Several risk factors may accelerate EHS development, but NSAIDs are

currently not considered a risk factor.

Adverse NSAID effects share a number of components with the dual EHS model –

GI damage, increased GI permeability, and inflammatory responses. Therefore, it can be

inferred that NSAIDs use could exacerbate EHS risk via these shared mechanisms. There

is limited research on NSAIDs and Tc during exercise and thermal stress. Existing

literature typically fails to measure inflammatory markers and GI damage, varies from low to high intensity exercise, and varies from temperate to hot environments. Furthermore, using ibuprofen or acetylsalicylate is practical, but neither drug is a potent COX inhibitor.

Naproxen is a commonly used, potent over-the-counter medication, but because it is relatively newer there is less research on its effects.

Physical conditioning, acclimatization, electrolyte and fluid balance, and other intrapersonal factors may impact Tc during activity. Considering existing literature, there is a clinically relevant question as to whether NSAIDs should also be considered a risk factor for EHS. Unfortunately, there is an overall lack of well-developed, controlled studies with intense exercise in thermal environments. Future research is needed to examine how

NSAIDs may negatively impact thermoregulation through effects on the GI tract and immune system.

METHODS

The purpose of this study is to determine the effects of over-the counter naproxen

on Tc, GI permeability, GI distress, and inflammatory markers in hydrated, cycling adults

in a thermal environment.

153

Research Design

We will utilize a double-blind, randomized, cross-over design to determine the

effects of naproxen on the dependent variables Tc, TNF-α, IL-6, GI permeability, GI

distress symptoms, occult fecal blood loss, P[Na+], HR, BP, and performance (distance

covered during a 10min time trial). Independent variables are naproxen (220mg

naproxen/dose), placebo (0mg naproxen/dose), thermal environment (35°C, 30%

humidity),279 and thermoneutral environment (15°C, 30% humidity).279 All participants

will attend an informational session and complete the STEEP test to estimate V̇ O2max.

Participants will be randomly assigned to 1 of 4 groups: 1) placebo and thermoneutral

(Control); 2) placebo and heat stress (PHeat); 3) naproxen and thermoneutral (Npx); and

4) naproxen and heat stress (NpxHeat) in a counterbalanced order with a minimum of 7

days between each data collection session. Dependent measures will be taken pre-, during,

and post- a 90 min cycling protocol.

Participants

A convenient sample of participants (n ≈ 20; age = 18-38 yrs) from the University

of South Carolina and surrounding community will be used to participate in this study. To

be included in the study participants must be moderately trained (male V̇ O2max between

280 35-40 mL/kg; female V̇ O2max between 32-40 mL/kg), non-heat acclimatized, in good

general health and will be accepted based upon the absence of cardiovascular, respiratory,

and metabolic disorders; musculoskeletal disorders preventing cycling exercise; fluid or

electrolyte balance disorders; and GI or swallowing disorders. Participants will be screened

using a Health and Injury History Questionnaire and read and sign an Institutional Review

Board approved Informed Consent Form prior to participation.

154

Experimental Conditions

Control Condition

Participants in the Control condition will report to the laboratory 24 hrs prior to data collection and will be given 3 placebo capsules (glucose) and a Take Home Instruction sheet. Participants will complete a 90 min cycle trial in a thermoneutral environment (15°C,

30% humidity).279

Placebo and Heat Stress (PHeat) Condition

Participants in the PHeat condition will report to the laboratory 24 hrs prior to data

collection and will be given 3 placebo capsules (glucose) and a Take Home Instruction

sheet. Participant will complete a 90 min cycle trial in a thermal environment (35°C, 30%

humidity).279

Naproxen (Npx) Condition

Participants in the Npx condition will report to the laboratory 24 hrs prior to data

collection and will be given 3 naproxen capsules (220mg naproxen/dose) and a Take Home

Instruction sheet. Participants will complete a 90 min cycle trial in a thermoneutral

environment (15°C, 30% humidity).279

Naproxen and Heat Stress (NpxHeat)

Participants in the NpxHeat condition will report to the laboratory 24 hrs prior to

data collection and will be given 3 naproxen capsules (220mg naproxen/dose) and a Take

Home Instruction sheet. Participant will complete a 90 min cycle trial in a thermal

environment (35°C, 30% humidity).279

155

Instruments and Protocols

Health and Injury History Questionnaire

The Health and Injury History Questionnaire references the Canadian Society for

Exercise Physiology, which developed the PAR-Q to identify individuals who should be seen by a medical doctor before becoming more physically active,281 and both the

American Medical Society for Sports Medicine and the American Orthopedic Society for

Sports Medicine.282 These two organizations have released pre-participation physical exam forms used to identify individuals at risk for injuries. Supplemental questions have been added to identify individuals with potential medical illness or injury (eg, GI disorders, history of EHS, metabolic disorders, daily NSAID use) that would exclude them from study participation.

Dosage for Experimental Conditions

A 24 hr dosage (placebo or naproxen) will be administered per the manufacturer’s recommendations of 1 capsule every 8 hrs, with the final dosage being taken upon arrival to the laboratory. A total of 3 capsules is consumed per the experimental protocol. Each dosage will be taken with 1 8oz glass of water and not with food, as this may alter the effectiveness of the dose. Previous studies have utilized NSAIDs to examine the effects of a 24 hrs dose on GI permeability14,260 and our total 24 hr dosage (660 mg) is comparable

to previous research.14,235,260,283

Take Home Instruction Packets

Upon arrival to the laboratory 24 hrs prior to data collection, participants will be

given a Pre-Data Collection Take Home Packet containing: 3 capsules (placebo or

naproxen) with directions for taking capsules, a fecal occult blood test kit with directions,

156

and a pre-data collection GI symptoms survey. After completing the data collection trial,

participants will be given a Post-Data Collection Take Home Packet containing: directions

for collecting the last fecal stool sample, a GI symptom survey to complete when collecting the last fecal sample, and directions on returning the kit and survey to the researchers.

STEEP Test

The STEEP Test, adapted from the Bruce Protocol for cyclist, will estimate

284 V̇ O2max. The test will begin at a low intensity and progressively increase the work load

15% every 1 minute. A V̇ O2max between 40-50 mL/kg in males and 32-40 mL/kg in females will be considered “trained” and qualify for participation in the study. Results from V̇ O2max

and HR will also be utilized to determine appropriate resistance for each participant to

279 maintain a steady state of 70% V̇ O2max during cycling exercise trial.

Cycle Exercise Trial

279 Participants will complete a 90 min cycle trial at a steady state of 70% V̇ O2max

for 80 min. The final 10 min of the trial will be completed at maximum effort. Distance

covered during the 10 min will be utilized to determine performance. Cycling was chosen

as the form of exercise due to the lesser impact on the GI tract compared to running.5,38

Any changes in the dependent variables is better explained from the effects of naproxen or

environment and not as responses to running. The exercise trial will begin with a 3 min

warm and be conducted in an environmental chamber.

Inflammatory Cytokines

An intravenous catheter will be placed in the cubital vein prior to exercise. The

catheter is secured so that only one needle stick is required and blood is collected pre-,

during, and immediately post-exercise. A total of 6 ml per blood draw will be collected

157

into an ethylenediaminetetraacetic acid (EDTA) vacutainer tube and inverted several times to mix. EDTA tubes are the preferred choice to prevent coagulation of the blood sample prior to analysis.285 TNF-α and IL-6 is assessed using enzyme linked immunosorbent assay

(ELISA) using high sensitivity kits (BD Biosciences TNF-α and IL-6 ELISA kits, BD

Biosciences, San Diego, CA).

Gastrointestinal Permeability

To determine GI permeability, participants will arrive to the laboratory, void their bladder and then ingest 150 ml of a solution containing 5 g sucrose, 5 g lactulose, and 2 g mannitol (Sigma-Aldrich Co. LLC, St. Louis, MO). Urinary excretion of sugars will be determined by urine sugar concentration, total urine volume, and dose of sugar ingested.286

Urine samples will be prepared and analyzed for percent excretion of sucrose, lactulose, and mannitol using gas chromatography with mass spectrometry.287 Sucrose will be used to determine gastroduodenal permeability, while the ratio of lactulose/mannitol is used to determine small intestine permeability.14

Fecal Occult Blood Test

To determine GI bleeding fecal occult blood is measured using take home kits

(Fisher HealthCareTM Sure-VueTM Fecal Occult Blood Slide Tests System, Thermo Fisher

Scientific Inc., Waltham, MA). Kits include triple slide monitors, collection tissues, applicators, test instructions, and a mailing envelope. For each experimental trial, participants will provide the first stool sample after initiating NSAID/placebo administration and the first stool sample following the exercise protocol. Participants are instructed to document time of stool sample collection.

158

Hydration and Sodium Balance

Participants will be required to be euhydrated prior to beginning data collection.

Hydration status is assessed by Posm, urine specific gravity (Usg), and body mass (BM).

Euhydration will be defined as a Posm < 290 mOsmols, Usg < 1.020, and/or %BM < 1%

change from baseline BM.288

Blood is obtained pre-, during, and immediately post-exercise. Blood samples are

collected into a 6ml EDTA vacutainer tube, centrifuged at 3000 rpm for 15 min, plasma

pipetted into microtubes, and stored at -20°C until analysis. Osmolality is measured using

a freeze point depression (Multi-sample Osmometer model 2020, Advanced Instruments,

Norwood, MA). Plasma osmolality is considered one of the most valid and reliable

measures of acute hydration status changes289 and is used to ensure participants are in a

hydrated state throughout each trial.

Urine is obtained pre- and post-exercise to obtain immediate estimates of hydration status. Participants must be in a euhydrated state prior to completing the data collection trial. A clean catch method will be used in which the participant is instructed to urinate a small amount before placing the specimen cup in midstream to collect a minimum of 1 ounce of urine. Usg is measured using a handheld clinical refractometer (model REF 312,

Atago Company Ltd., Tokyo, Japan). Designed for clinical use, the clinical refractometer measures total concentration of liquid compared to water. The clinical refractometer is calibrated before and after each Usg measurement using two drops of distilled water on the prism, holding the instrument toward light, and adjusting the turning screw to correspond the light and dark boundary at 1.000. Following calibration, two drops of specimen are pipetted onto the dry prism, the refractometer is held to the light, and the Usg is recorded.290

159

A Tanita body composition analyzer (model TBF-300, Tanita Corporation,

Arlington Heights, IL) is used to measure BM pre-, immediately post-exercise, and 3 hrs post-exercise. Participants are asked to void urine before stepping on scale, dress in shorts and t-shirt, and towel off any sweat. Baseline BM measures are taken at the information session, where participants will provide a urine sample to determine euhydration (Usg <

1.020).

® To determine changes in P[Na+] we will utilize ion-selective electrodes (EasyLyte

Na/K electrolyte analyzer model REF 2277, Medica, Bedford, MA). Plasma will be

obtained using an intravenous catheter placed in the cubital vein. Blood will be centrifuged,

plasma pipetted into microtubes and stored at -20°C until analysis. All samples will be

discarded after analysis.

Core Body Temperature

Tc is recorded continuously throughout the exercise trial using rectal temperature

(model 4600, YSI Precision Temperature Group, Dayton, OH). Participants are not allowed

to exceed a Tc > 40°C/104°F. Rectal temperature is accepted as a valid and reliable

measure of Tc,291 frequently used as a criterion measure of Tc during exercise under

thermal stress,292,293 and is used in studies examining GI permeability and aspirin.14,15

Gastrointestinal Symptom Index

Gastrointestinal distress will be assessed using a symptom questionnaire adopted from previous research.45,294 The index is divided into 3 sections: 1) upper abdominal

problems (heart burn, reflux, belching, bloating, stomach pain/cramping, nausea,

vomiting); 2) lower abdominal problems (intestinal/lower abdominal pain/cramping,

flatulence, urge to defecate, side aches/stitch, loose stoole, diarrhea); and 3) systemic

160

problems (dizziness, headache, muscle cramps, urge to urinate).45 Symptoms are scored on

a 10-point scale (0 = no problems at all and 9 = the worst it has ever been). A score of > 4

is considered “serious”.294 Questionnaires will be administered pre-, immediately post-, and

2 hrs post-exercise. To determine GI symptoms during exercise, participants are verbally asked about current GI symptoms and severity every 15 min.

Rate of Perceived Exertion

The Borg Scale is used to measure the participants’ rate of RPE pre-, every 15 min during, and immediately post-exercise.

Cardiovascular

To ensure participants remain at safe limits and determine any effects from NSAID use, HR is continuously monitored using Polar HR monitors (Polar Electro Inc., Lake

Success, NY). In addition, BP is measured every 15 min to ensure participants maintain a normal BP response throughout the trial.

Diet and Activity Logs

Participants will be required to track diet and activity for 3 days prior to and 1 day after data collection using an online nutrition software (ESHA Research, Salem, OR).

Participants are asked to refrain from red meat for 3 days prior and 1 day after data collection to limit false positive fecal occult blood tests. Logs are also used to help participants maintain similar dietary and activity habits during the 24 hrs prior to data collection.

161

Experimental Procedures (Figure 5.2)

Information Session

Consenting participants will sign the informed consent form and complete the

Health and Injury Questionnaire. If participant has no disqualifying conditions (i.e. metabolic, cardiovascular, respiratory, and/or musculoskeletal disorders; current pain medication or NSAID use, etc.) he/she will complete the STEEP test to measure V̇ O2max.

Participants are randomly assigned to 1 of 4 groups, with all 4 groups being completed in a randomized cross-over design. Both the participant and primary investigator are blind to whether the participant is completing the Npx or placebo group. Take Home Instructions are reviewed with the participant at the end of a session and prior to leaving the laboratory.

Participants are not allowed to take any other medications for pain or inflammation during the course of the study. Participants are instructed to refrain from intense, vigorous exercise

2 days (48 hrs) prior to data collection, no exercise 24 hrs prior to data collection, and will complete an online dietary and activity log from 72 hrs prior to and 24 hrs post data collection.172

Data Collection Session

Participants will report to the laboratory 24 hrs prior to data collection to obtain 3 pills (Npx or placebo), fecal occult blood tests, and Take Home Instructions. Participants will consume the 3 pills over the 24 hr period, drinking 1 8oz glass of water with each dose and not with food. At least 2 hrs prior to arriving for data collection, participants should consume a small meal and are instructed to consume the same meal prior to each trial. All data collection sessions are conducted at the same time of day for each participant and separated by a minimum of 7 days.

162

Upon arrival to the laboratory, participants will consume the 3rd pill. A member of

the research team will measure hydration status to determine if the participant is in a

euhydrated state. Participants are provided a rectal thermometer probe and a private area

to insert the probe and a baseline Tc will be taken. A venous catheter is inserted into the

cubital vein and 2 6ml vials of blood will be collected for baseline measures. Participants

will complete a 3 min warm up on the bike prior to starting the 90 min cycle protocol.

Participants will consume 3.5ml/kg of water every 15 min to maintain hydration status. Tc and HR will be monitored continuously. RPE and BP will be measured every 15 min. Two

6ml vials of blood will be taken pre-, 80 min, immediately post-cycle, and 3 hours post-

cycle. At the conclusion of the cycling protocol, participants will weigh, provide a urine

sample, and complete the GI symptom index questionnaire. Participants will then rest for

3 hrs in semi-reclined/seated position in a thermoneutral environment. Participants will

consume 3.5ml/kg of water every 15 min to maintain hydration status. Blood, urine,

weight, and GI symptom measures are taken at the conclusion of 3 hrs rest. The cycling

protocol will cease if one of the following criteria is met: 1) participant completes the 90

min cycle protocol; 2) participant’s Tc = 40°C; 3) participant experiences severe GI distress

(eg, vomiting); or 4) participant requests to stop the trial.

Statistical Analysis

SPSS statistical software (version XIIV; SPSS Inc, Chicago, IL) is used for all

analyses. Descriptive statistics (mean and standard deviations) for all dependent variables

is calculated. Repeated measures ANOVA is used to determine the differences of all

dependent variables during exercise between the 4 groups. Significance level is set at P <

163

.05 for all analyses. Power analysis, a priori, calculated a participant number of 20 to have

a power of 0.90, which is comparable to previously published studies.295

REFERENCES

1. McArdle WD, Katch FI, Katch VL. Exercise Physiology: Energy, Nutrition, and Human Performance. Fifth ed. Baltimore, MD: Lippincott Williams & Wilkins; 2001.

2. Berg A, Muller H-M, Rathmann S, Deibert P. The gastrointestinal system - an essential target organ of the athlete's health and physical performance. Exerc Immunol Rev. 1999;5:78-95.

3. Granger DN, Richardson PDI, Kvietys PR, Mortillaro NA. Intestinal blood flow. Gastroenterol. 1980;78(4):837-863.

4. Knochel JP. Enivonrmental heat illness: an eclectic review. Arch Intern Med. 1974;133:841-864.

5. Lim CL, Mackinnon LT. The roles of exercise-induced immune system disturbances in the pathology of heat stroke : the dual pathway model of heat stroke. Sports Med. 2006;36(1):39-64.

6. Armstrong LE, Johnson EC, Casa DJ, et al. The American football uniform: uncompensable heat stress and hyperthermic exhaustion. J Athl Train. 2010;45(2):11.

7. Epstein Y, Shapiro Y, Brill S. Role of surface area-to-mass ratio and work efficiency in heat intolerance. J Appl Physiol. 1983;54(3):831-836.

8. Casa DJ. Exercise in the heat. II. Critical concepts in rehydration, exertional heat illnesses, and maximizing athletic performance. J Athl Train. 1999;34(3):253-262.

9. Binkley HM, Beckett J, Casa DJ, Kleiner DM, Plummer PE. National Athletic Trainers' Association position statement: exertional heat illnesses. J Athl Train. 2002;37(3):329-343.

10. Proulx CI, Ducharme MB, Kenny GP. Safe cooling limits from exercise-induced hyperthermia. Eur J Appl Physiol. 2006;96(4):434-445.

11. Clements JM, Casa DJ, Knight JC, et al. Ice-water immersion and cold-water immersion provide similar cooling rates in runners with exercise-induced hyperthermia. J Athl Train. 2002;37(2):146-150.

12. Brooks M. Ten years after Korey Stringer's death, heat-related deaths still plague football teams around the country. The Washington Post. August 12, 2011, 2011.

164

13. Ryan AJ, Chang R-T, Gisolfi CV. Gastrointestinal permeability following aspirin intake and prolonged running. Med Sci Sports Exerc. 1996;28(6):698-705.

14. Lambert GP, Boylan M, Laventure JP, Bull A, Lanspa S. Effect of aspirin and ibuprofen on GI permeability during exercise. Int J Sports Med. 2007;28(9):722-726.

15. Lambert GP, Broussard LJ, Mason BL, Mauermann WJ, Gisolfi CV. Gastrointestinal permeability during exercise: effects of aspirin and energy-containing beverages. J Appl Physiol. 2001;90(6):2075-2080.

16. Darling RL, Romero JJ, Dial EJ, Akunda JK, Langenbach R, Lichtenberger LM. The effects of aspirin on gastric mucosal integrity, surface hydrophobicity, and prostaglandin metabolism in cyclooxygenase knockout mice. Gastroenterology. 2004;127(1):94-104.

17. Laporte J-R, Ibanez L, Vidal X, Vendrell L, Leone R. Upper gastrointestinal bleeding associated with the use of NSAIDs: newer versus older agents. Drug Saf. 2004;27(6):411-420.

18. Wallace JL. Prostaglandins, NSAIDs, and gastric mucosal protection: why doesn't the stomach digest itself? Physiol Rev. 2008;88:1547-1565.

19. Marieb EN. Human Anatomy and Physiology. 5th ed. San Francisco, CA: Benjamin Cummings; 2001.

20. Hadi NA, Giouvanoudi A, Morton R, Horton PW, Spyrou NM. Variations in gastric emptying times of three stomach regions for simple and complex meals using scintigraphy IEEE Transactions on Nuclear Science. 2002;49(5):2328-.

21. Berne RM, Levy MN. Principles of Physiology. 3rd ed. St. Louis, MO: Mosby, Inc; 2000.

22. Sears CL. A dynamic partnership: celebrating our gut flora. Anaerobe. 2005;11(5):247-251.

23. ter Steege RW, Kolkman JJ. Review article: the pathophysiology and management of gastrointestinal symptoms during physical exercise, and the role of splanchnic blood flow. Aliment Pharmacol Ther. 2012;35(5):516-528.

24. Rehrer N, Smets A, Reynaert H, Goes E, de Meirleir K. Effect of exercise on portal vein blood flow in man. Med Sci Sport Exerc. 2001;33(9):1533-1537.

25. Constanti A, Bartke A, Khardori R. Basic Endocrinology. Amsterdam, Netherlands: Harwood Academic Publishers; 1998.

26. Lambert GP. Stress-induced gastrointestinal barrier dysfunction and its inflammatory effects. J Anim Sci. 2009;87(14 Suppl):E101-E108.

165

27. Turner JR. Intestinal mucosal barrier function in health and disease. Nat Rev Immunol. 2009;9(11):799-809.

28. Lambert GP. Role of gastrointestinal permeability in exertional heatstroke. Exerc Sport Sci Rev. 2004;32(4):185-190.

29. Nusrat A, Turner JR, Madara JL. Molecular physiology and pathophysiology of tight junctions IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Am J Physiol Gastrointest Liver Physiol. 2000;279:G851-G857.

30. Turner JR. 'Putting the squeeze' on the tight junction: understanding cytoskeletal regulation. Semin Cell Dev Biol. 2000;11(4):301-308.

31. Turner JR, Rill BK, Carlson SL, et al. Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation. Am J Physiol Cell Physiol. 1997;273:C1378-C1385.

32. Berglund JJ, Riegler M, Zolotarevsky Y, Wenzl E, Turner JR. Regulation of human jejunal transmucosal resistance and MLC phosphorylation by Na+-glucose cotransport. Am J Physiol Gastrointest Liver Physiol. 2001;281:G1487-G1493.

33. Markov AG, Veshnyakova A, Fromm M, Amasheh M, Amasheh S. Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine. J Comp Physiol B. 2010;180(4):591-598.

34. Goodman CC, Fuller KS. Pathology: Implications for the Physical Therapist. 3rd ed. St. Louis, MO: Saunders Elsevier; 2009.

35. Moses FM. The effect of exercise on the gastrointestinal tract. Sports Med. 1990;9(3):159-172.

36. Peters HPF, Bos M, Seebregts L, et al. Gastrointestinal symptoms in long-distance runners, cyclists, and triathletes: prevalence, medication, and etiology. Am J Gastroenterol. 1999;94(6):1570-1581.

37. Waterman JJ, Kapur R. Upper gastrointestinal issues in athletes. Curr Sports Med Rep. 2012;11(2):99-104.

38. Rehrer NJ, Meijer GA. Biomechanical vibration of the abdominal region during running and bicycling. J Sports Med Phys Fitness. 1991;31(2):231-234.

39. Chen L, Yang G, Grosser T. Prostanoids and inflammatory pain. Prostaglandins Other Lipid Mediat. 2013;104-105:58-66.

40. Bruggeman TM, Wood JG, Davenport HW. Local control of blood flow in the dog's stomach: vasodilation caused by acid back diffusion following topical application of salicylic acid. Gastroenterol. 1979;77:736-744.

166

41. Carden DL, Granger DN. Pathophysiology of ischemia-reperfusion injury. J Pathol. 2000;190:255-266.

42. Collard CD, Gelman S. Pathophysiology, clinical manifestations, and prevention of ischemia–reperfusion injury. Anesthesiology. 2001;94:1133-1138.

43. Peters HPF, Wiersma WC, Koerselman J, et al. The effect of a sports drink on gastroesophageal reflux during a run-bike-run test. Int J Sports Med. 1999;20:65-70.

44. van Nieuwenhoven MA, Brouns F, Brummer RJ. The effect of physical exercise on parameters of gastorintestinal function. Neurogastroenterol Mot. 1999;11:431-439.

45. Pfeiffer B, Cotterill A, Grathwohl D, Stellingwerff T, Jeukendrup AE. The effect of carbohydrate gels on gastrointestinal tolerance during a 16-km run. Int J Sport Nutr Exerc Metab. 2009;19:485-503.

46. McCabe III ME, Peura DA, Kadakia SC, Bocek Z, Johnson LF. Gastrointestinal blood loss associated with running a marathon. Digest Dis Sci. 1986;31(11).

47. Rehrer NJ, Beckers EJ, Brouns F, Ten Hoor F, Saris WHM. Effects of dehydration on gastric emptying and gastrointestinal distress while running. Med Sci Sports Exerc. 1990;22(6):790-795.

48. Collings KL, Pierce Pratt F, Rodriguez-Stanley S, Bemben M, Miner PB. Esophageal reflux in conditioned runners, cyclists, and weightlifters. Med Sci Sports Exerc. 2003;35(5):730-735.

49. Stuempfle KJ, Hoffman MD, Hew-Butler T. Association of gastrointestinal distress in ultramarathoners with race diet. Int J Sport Nutr Exerc Metab. 2013;23:103-109.

50. Oort FA, Terhaar Sive Droste JS, Van Der Hulst RW, et al. Colonoscopy-controlled intra-individual comparisons to screen relevant neoplasia: faecal immunochemical test vs. guaiac-based faecal occult blood test. Aliment Pharmacol Ther. 2010;31(3):432-439.

51. Young GP, Cole S. New stool screening tests for colorectal cancer. Digestion. 2007;76(1):26-33.

52. Young GP, St. John DJB, Winawer SJ, Rozen P. Choice of fecal occult blood tests for colorectal cancer screening: recommendations based on performance characteristics in population studies. Am J Gastroenterol. 2002;97(10):2499-2507.

53. Bjarnason I, Peters TJ, Levi AJ. Intestinal permeability: clinical correlates. Dig Dis. 1986;4(2):83-92.

54. Berkes J, Viswanathan VK, Savkovic SD, Hecht G. Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation. Gut. 2003;52:439-451.

167

55. Baert FJ, R. DHG, Peeters M, et al. Tumour necrosis factor a antibody (infliximab) therapy profoundly downregulates the inflammation in Chrohn's ileocolitis. Gastroenterol. 1999;116:22-28.

56. Suenaert P, Bulteel V, Lemmens L, et al. Anti-tumour necrosis factor treatment restores the gut barrier in Chohn's disease. Am J Gastroenterol. 2002;97(8):2000- 2004.

57. Tazuke Y, Drongowski RA, Teitelbaum DH, Coran AG. Interleukin-6 changes tight junction permeability and intracellular phospholipid content in a human enterocyte cell culture model. Pediatr Surg Int. 2003;19(5):321-325.

58. Madara JL. Migration of neutrophils through epithelial monolayers. Trends Cell Bio. 1994;4(1):4-7.

59. Voet D, Voet JG. Biochemistry. Hoboken, NJ: John Wiley & Sons, Inc.; 2011.

60. Nelson DL, Cox MM. Lehninger Principles of Biochemistry. 5th ed. New York, NY: W. H. Freeman and Company; 2008.

61. Brock-Utne JG, Gaffin SL, Wells MT, et al. Endotoxaemia in exhausted runners after a long-distance race. S Afr Med J. 1988;73(9):533-536.

62. Rao R. Endotoxemia and gut barrier dysfunction in alcoholic liver disease. Hepatology. 2009;50(2):638-644.

63. Doran JE. Biological effects of endotoxin. In: Kraft CH, ed. Gut-derived infectious toxic shock: current studies in hematology and blood transfusion. Vol 59: Basel Krager; 1992:66-99.

64. Mimura Y, Sakisaka S, Harada M, Sata M, Tanikawa K. Role of hepatocytes in direct clearance of lipopolysaccharide in rats. Gastroenterol. 1995;109:1969-1976.

65. Gazzano-Santoro H, Parent JB, Grinna L, et al. High-affinity binding of the bactericidal/permeability-increasing protein and a recombinant amino-termial fragment to the lipid A region of lipopolysaccharide. Infect Immun. 1992;60(11):4754-5761.

66. Mannion BA, Weiss J, Elsbach P. Separation of sublethal and lethal effects of the bactericidal/permeability increasing protein on Escherichia coli. J Clin Invest. 1990;85(3):853-860.

67. Canny G, Levy O. Bactericidal/permeability-increasing protein (BPI) and BPI homologs at mucosal sites. Trends Immunol. 2008;29(11):541-547.

68. Maxton DG, Bjarnason I, Reynolds AP, Catt SD, Peters TJ, Menzies IS. Lactulose 51Cr-labelled ethylenediaminetetra-acetate, L-rhamnose and polyethyleneglycol 500

168

as probe markers for assessment in vivo of human intestinal permeability. Clin Sci. 1986;71:71-80.

69. Davies NM. Review article: non-steroidal anti-inflammatory drug-induced gastrointestinal permeability. Aliment Pharmacol Ther. 1998;12:303-320.

70. Ukabam SO, Cooper BT. Small intestinal permeability to mannitol, lactulose, and polyethylene glycol 400 in celiac disease. Dig Dis Sci. 1984;29:809-816.

71. Zaslavsky BY, Baevskii AV, Rogozhin SV, et al. Relative hydrophobicity of synthetic macromolecules. I. Polyethylene glycol, polyacrylamide, and polypyrrolidone J Chromatogr. 1985;285:63-68.

72. Ashton T, Young I, Davison G, et al. Exercise-induced endotoxemia: the effect of ascorbic acid supplementation. Free Radical Bio Med. 2003;35(3):284-291.

73. Camus G, Nys M, Poortmans JR, et al. Endotoxemia, production of tumor necrosis factor-a and polymorphonuclear neutrophil activation following strenous exercise in humans. Eur J Appl Physiol. 1998;79:62-68.

74. Jeukendrup AE, Vet-Joop K, Sturk A, et al. Relationship between gastro-intestinal complaints and endotoxaemia, cytokine release and the acute-phase reaction during and after a long-distance triathlon in highly trained men. Clin Sci. 2000;98:47-55.

75. Hurley JC. Endotoxemia: methods of detection and clinical correlates. Clin Microbiol Rev. 1995;8(2):268-292.

76. Li H, Edin ML, Gruzdev A, et al. Regulation of T helper cell subsets by cyclooxygenases and their metabolites. Prostaglandins Other Lipid Mediat. 2013;104-105:74-83.

77. Walch M, Dotiwala F, Mulik S, et al. Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes. Cell. 2014;157(6):1309-1323.

78. Grazia Roncarolo M, Gregori S, Battaglia M, Bacchetta R, Fleischhauer K, Levings MK. Interleukin-10-secreting type 1 regulatory T cells in rodents and humans. Immunol Rev. 2006;212:28-50.

79. Kondělková K, Vokurková D, Krejsek J, Borská L, Fiala Z, Andrýs C. Regulatory T cells (Treg) and their roles in immune system with respect to immunopathological disorders Acta Medica. 2010;53(2):73-77.

80. Grossman WJ, Verbsky JW, Barchet W, Colonna M, Atkinson JP, Ley TJ. Human T regulatory cells can use the perforin pathway to cause autologous target cell death. Immunity. 2004;21(4):589-601.

81. Facciabene A, Motz GT, Coukos G. T-regulatory cells: key players in tumor immune escape and angiogenesis. Cancer Res. 2012;72(9):2162-2171.

169

82. Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol. 2008;9(5):503-510.

83. Vivier E, Raulet DH, Moretta A, et al. Innate or adaptive immunity? The example of natural killer cells. Science. 2011;331(6013):44-49.

84. Bouchama A, Parhar RS, El-Yazigi A, Sheth K, Al-Sedairy S. Endotoxemia and release of tumor necrosis factor and interleukin Ia in acute heatstroke. J Appl Physiol. 1991;70(6):2640-2644.

85. Brocker C, Thompson D, Matsumoto A, Nebert DW, Vasiliou V. Evolutionary divergence and functions of the human interleukin (IL) gene family. Hum Genom. 2010;5(1):30-55.

86. Commins SP, Borish L, Steinke JW. Immunologic messenger molecules: cytokines, interferons, and chemokines. J Allergy Clin Immunol. 2010;125(2 Suppl 2):S53-S72.

87. Akira S, Hirano T, Taga T, Kishimoto T. Biology of multifunctional cytokines: IL 6 and related molecules (IL 1 and TNF). FASEB J. 1990;4:2860-2867.

88. Del Prete G, De Carli M, Almerigogna F, Grazia Giudizi M, Biagiotti R, Romagnani S. Human IL-10 is produced by both type 1 helper (Thl) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production. 1993;150(2):353-360.

89. Ding L, Linsley PS, Haung LY, Germain RN, Shevach EM. IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression. J Immunol. 1993;151(3):1224-1234.

90. Zenewicz LA, Flavell RA. Recent advances in IL-22 biology. Int Immunol. 2011;23(3):159-163.

91. Beutler B, Cerami A. The biology of cachectin/TNF - a primary mediator of the host response. Ann Rev Immunol. 1989;7:625-655.

92. Tracey KJ, Fong Y, Hesse DG, et al. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature. 1987;330:662-664.

93. Santoro MG. Heat shock factors and the control of the stress response. Biochem Pharmcol. 2000;59(1):55-63.

94. Musch MW, Ciancio MJ, Sarge K, Chang EB. Induction of heat shock protein 70 protects intestinal epithelial IEC-18 cells from oxidant and thermal injury. Am J Physiol Cell Physiol. 1996;270:C429-C436.

95. Welch WJ. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol Rev. 1992;72(4):1063- 1081.

170

96. Kregel KC. Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J Appl Phys. 2002;92(5):2177-2186.

97. Hightower LE. Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides. J Cell Physiol. 1980;102:407-427.

98. Nickells RW, Browder LW. A role for glyceraldehyde-3-phosphate dehydrogenase in the development of thermotolerance in Xenopus laevis embryos. Dev Biol. 1985;112:391-395.

99. Welch WJ, Suhan JP. Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment. J Cell Biol. 1985;101(4):1198-1211.

100. Rubin GM, Hogness DS. Effect of heat shock on the synthesis of low molecular weight RNAs in Drosophila: accumulation of a novel form of 5S RNA. Cell. 1975;6:207-213.

101. Polla BS, Bachelet M, Elia G, Santoro MG. Stress proteins in inflammation. Ann N Y Acad Sci. 1998;851(1):75-85.

102. Todryk S, Melcher AA, Hardwick N, et al. Heat shock protein 70 induced during tumor cell killing induces Th1 cytokines and targets immature dendritic cell precursors to enhance antigen uptake. J Immunol. 1999;163:1398-1408.

103. Jaattela M, Wissing D. Heat-shock proteins protect cells from monocyte cytotoxicity: possible mechanism of self-protection. J Exp Med. 1993;177:231-236.

104. Muller E, Munker R, Issels R, Wilmanns W. Interaction between tumor necrosis factor-a and HSP70 in human leukemia cells. Leukemia Res. 1993;17(6):523-526.

105. Ensor JE, Wiener SM, McCrea KA, Viscardi RM, Crawford EK, Hasday JD. Differential effects of hyperthermia on macrophage interleukin-6 and tumor necrosis factor-alpha expression. Am J Physiol Cell Physiol. 1994;266:C967-C974.

106. Snyder YM, Guthrie L, Evans GF, Zuckerman SH. Transcriptional inhibition of endotoxin-induced monokine synthesis following heat shock in murine peritoneal macrophages. J Leukoc Biol. 1992;51:181-187.

107. Landry J, Bernier D, Chretien P, Nicole LM, Tanguay RM, Marceau N. Synthesis and degradation of heat shock proteins during development and decay of thermotolerance. Cancer Res. 1982;42:2457-2461.

108. O'Neill S, Ingman TG, Wigmore SJ, Harrison EM, Bellamy CO. Differential expression of heat shock proteins in healthy and diseased human renal allografts. Ann Transplant. 2013;18:550-557.

171

109. Samali A, Cotter TG. Heat shock proteins increase resistance to apoptosis. Exp Cell Res. 1996;223:163-170.

110. Mosser DD, Martin LH. Induced thermotolerance to apoptosis in human T lymphocyte cell line. J Cell Physiol. 1992;151:561-570.

111. Toyokuni S. Reactive oxygen species-induced molecular damage and its application in pathology. Pathol Int. 1999;49:91-102.

112. Jacquier-Sarlin MR, Polla BS. Dual regulation of heat-shock transcription factor (HSF) activation and DNA-binding activity by H2O2: role of thioredoxin. Biochem J. 1996;318(187-193).

113. Ramwell PW, Leovey EMK, Sintetos AL. Regulation of the arachidonic acid cascade. Bio Reprod. 1977;16:70-87.

114. Moncada S, Vane JR. Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane A2, and prostacyclin. Pharmacol Rev. 1978;30(3):293- 331.

115. Straus DS, Glass CK. Cyclopentenone prostaglandins: new insights on biological activities and cellular targets. Med Res Rev. 2001;21(3):185-210.

116. Brock TG, McNish RW, Peters-Golden M. Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2. J Biol Chem. 1999;274(17):11660-11666.

117. Prostanoids - prostaglandins, prostacyclins, and thromboxanes. AOCS Lipid Library; 2014. Accessed 5/12/2015.

118. Garella S, Matarese RA. Renal effects of prostaglandins and clinical adverse effects of nonsteroidal anti-inflammatory agents. Medicine. 1984;63(3):165-181.

119. McGiff JC, Crowshaw K, Terragno NA, Lonigro AJ. Release of a prostaglandin-like substance into renal venous blood in response to angiotensin II. Cir Res. 1970;26:121-130.

120. Dunn MJ, Greely HP, Valtin H, Kintner LB, Beeuwkes R. Renal excretion of prostaglandins E2 and F2alpha in diabetes insipidus rats. Am J Physiol. 1978;235(6):E624-E627.

121. McGiff JC, Terragno NA, Malik KU, Lonigro AJ. Release of a prostaglandin E-like substance from canine kidney by bradykinin. Cir Res. 1972;31:36-43.

122. Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol. 2011;31(5):986-1000.

172

123. Morita I. Distinct functions of COX-1 and COX-2. Prostaglandins Other Lipid Mediat. 2002;68-69:165-175.

124. Santoro MG, Garaci E, Amici C. Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells. Proc Natl Acad Sci U S A. 1989;86(21):8407-8411.

125. Kim H-S, Rhim H, Jeong S-W, Kim JW, Kim I-K. Induction of apoptosis dependent on caspase activities and growth arrest in HL-60 cells by PGA2. Prostaglandins Other Lipid Mediat. 2002;70:169-183.

126. Santoro MG. Antiviral activity of cyclopentenone prostanoids. Trends Microbiol. 1997;5(7):276-281.

127. Dusting GJ, Moncada S, Vane JR. Disappearance of prostacyclin (PGI2) in the circulation of the dog [proceedings]. Br J Pharmacol. 1978;62(3):414P-415P.

128. Gretzer B, Maricic N, Respondek M, Schuligoi R, Peskar B. Effects of specific inhibition of cyclo-oxygenase-1 and cyclo-oxygenase-2 in the rat stomach with normal mucosa and after acid challenge. Br J Pharmacol. 2001;132:1565-1573.

129. Ferraz J-GP, McKnight W, Sharkey KA, Wallace JL. Impaired vasodilatory responses in the gastric microcirculation of anesthetized rats with secondary biliary cirrhosis. Gastroenterol. 1995;108:1183-1191.

130. Tsutsumi S, Haruna R, Tomisato W, et al. Effects of prostaglandins on spontaneous apoptosis in gastric mucosal cells. Dig Dis Sci. 2002;47(1):84-89.

131. Hoshino T, Tsutsumi S, Tomisato W, Hwang HJ, Tsuchiya T, Mizushima T. Prostaglandin E2 protects gastric mucosal cells from apoptosis via EP2 and EP4 receptor activation. J Biol Chem. 2003;278(15):12752-12758.

132. Befrits R, Johannsen C. Oral PGE2 inhibits gastric acid secretion in man. Prostaglandins. 1985;29(1):143-152.

133. Graham DY, Agrawal NM, Campbell DR, et al. Ulcer prevention in long-term users of nonsteroidal anti-inflammatory drugs. Results of a double-blind, randomized, multicenter, active- and placebo-controlled study of misoprostol vs lansoprazole. Arch Intern Med. 2002;162:169-175.

134. Konturek SJ, Robert A, Hanchar AJ, Nezamis JE. Comparison of prostacyclin and prostaglandin E2 on gastric secretion, gastrin release, and mucosal blood flow in dogs. Dig Dis Sci. 1980;25(9):673-679.

135. Way L, Durbin RP. Inhibition of gastric acid secretion in vitro by prostaglandin E1. Nature. 1969;221:874-875.

173

136. Soll AH. Specific inhibition by prostaglandins E2 and 12 of histamine-stimulated [14C]aminopyrine accumulation and cyclic adenosine monophosphate generation by isolated canine parietal cells. J Clin Invest. 1980;65:1222-1229.

137. Bandyopadhyay B, Bandyopadhyay SK. Role of prostaglandin in the regulation of gastric H+ transporting system. Indian J Clin Biochem. 1998;13(1):41-45.

138. Domschke W, Domschke S, Hornig D, Demling L. Prostaglandin-stimulated gastric mucus secretion in man. Acta Hepto-Gastroenterol. 1978;25:292-294.

139. Horton EW, Main IHM, Thompson CJ, Wright PM. Effect of orally administered prostaglandin E1 on gastric secretion and gastrointestinal motility in man. Gut. 1968;9:655-658.

140. Karim SMM, Carter DC, Bhana D, Adaikan Ganesan P. Effect of orally administered prostaglandin E2 15-methyl analogues on gastric secretion. Br Med J. 1973;1:143- 146.

141. Kauffman GLJ, Reeve JJJ, Grossman MI. Gastric bicarbonate secretion: effect of topical and intravenous 16,16-dimethyl prostaglandin E2. Am J Physiol Gastrointest Liver Physiol. 1980;239(1):G44-G48.

142. Keogh JP, Allen A, Garner A. Relationship between gastric mucus synthesis, secretion and surface gel erosion measured in amphibian stomach in vitro E. Clin Exp Pharmacol Physiol. 1997;24:844-849.

143. Takahashi S, Takeuchi K, Okabe S. EP4 receptor mediation of prostaglandin E2- stimulated mucus secretion by rabbit gastric epithelial cells. Biochem Pharmcol. 1999;58:1997-2002.

144. McQueen S, Hutton D, Allen A, Garner A. Gastric and duodenal surface mucus gel thickness in rat: effects of prostaglandins and damaging agents. Am J Physiol Gastrointest Liver Physiol. 1983;245:G388-G393.

145. Takeuchi K, Kita K, Hayashi S, Aihara E. Regulatory mechanism of duodenal bicarbonate secretion: Roles of endogenous prostaglandins and nitric oxide. Pharmacol Ther. 2011;130(1):59-70.

146. Takeuchi K, Ueshima K, Hironaka Y, Fujiokaa Y, Matsumoto J, Okabe S. Oxygen free radicals and lipid peroxidation in the pathogenesis of gastric mucosal lesions induced by indomethacin in rats. Digestion. 1991;49(3):175-784.

147. Mersereau WA, Hinchey EJ. Role of gastric mucosal folds in formation of focal ulcers in the rat. Surgery. 1982;91(2):150-155.

148. Takeuchi K. Pathogenesis of NSAID-induced gastric damage: importance of cyclooxygenase inhibition and gastric hypermotility. World J Gastroenterol. 2012;18(18):2147-2160.

174

149. Grisham MB, Hernandez LA, Granger DN. Xanthine oxidase and neutrophil infiltration in intestinal ischemia. Am J Physiol Gastrointest Liver Physiol. 1986;251:G567-G574.

150. Wallace JL, Granger DN. Pathogenesis of NSAID gastropathy: are neutrophils the culprits? Trends Pharmacol Sci. 1992;13(4):129-131.

151. Ueki S, Takeuchi K, Okabe S. Gastric motility is an important factor in the pathogenesis of indomethacin-induced gastric mucosal lesions in rats. Dig Dis Sci. 1988;33(2):209-216.

152. Suzuki K, Araki H, Mizoguchi H, Furukawa O, Takeuchi K. Prostaglandin E inhibits indomethacin-induced gastric lesions through EP-1 receptors. Digestion. 2001;63:92- 101.

153. Gryglewski RJ, Szczeklik A, Wandzilak M. The effect of six prostaglandins, prostacyclin and iloprost on generation of superoxide anions by human polymorphonuclear leukocytes stimulated by zymosan or formyl-methionyl-leucyl- phenylalanine. Biochem Pharmcol. 1987;36(24):4209-4213.

154. Parks DA, Bulkley GB, Granger DN. Role of oxygen-derived free radicals in digestive tract diseases. Surgery. 1983;94(3):415-422.

155. Zushi S, Shinomura Y, Kiyohara T, et al. Role of prostaglandins in intestinal epithelial restitution stimulated by growth factors. Am J Physiol Gastrointest Liver Physiol. 1996;270:G757-G762.

156. Erickson RA, Tarnawski A, Dines G, Stachura J. 16,16-Dimethyl prostaglandin E2 induces villus contraction in rats without affecting intestinal restitution. Gastroenterol. 1990;99(3).

157. Gookin JL, Galanko JA, Blikslager AT, Argenzio RA. PG-mediated closure of paracellular pathway and not restitution is the primary determinant of barrier recovery in acutely injured porcine ileum. Am J Physiol Gastrointest Liver Physiol. 2003;285:G967-G979.

158. Blikslager AT, Roberts MC, Rhoads JM, Argenzio RA. Prostaglandins I2 and E2 have a synergistic role in rescuing epithelial barrier function in porcine ileum. J Clin Invest. 1997;100(8):1928-1933.

159. Calabresi L. High-density lipoproteins protect isolated rat hearts from ischemia- reperfusion injury by reducing cardiac tumor necrosis factor-alpha content and enhancing prostaglandin release. Cir Res. 2002;92(3):330-337.

160. Kawabe J, Ushikubi F, Hasebe N. Prostacyclin in vascular diseases. Circ J. 2010;74(5):836-843.

175

161. Hassid A, Dunn MJ. Microsomal prostaglandin biosynthesis of human kidney. J Biol Chem. 1980;255(6):2472-2475.

162. Stokes JB. Integrated actions of renal medullary prostaglandins in the control of water excretion. Am J Physiol Renal Fluid Electrolyte Physiol. 1981;240:F471-F480.

163. Dunn MJ, Zambraski EJ. Renal effects of drugs that inhibit prostagiandin synthesis. Kidney Int. 1980;18:609-622.

164. Flores D, Liu Y, Liu W, Satlin LM, Rohatgi R. Flow-induced prostaglandin E2 release regulates Na and K transport in the collecting duct. Am J Physiol Renal Physiol. 2012;303:F632-F638.

165. Boone M, Deen PM. Physiology and pathophysiology of the vasopressin-regulated renal water reabsorption. Pflugers Arch. 2008;456(6):1005-1024.

166. Aronoff DM, Neilson EG. Antipyretics: mechanisms of action and clinical use in fever suppression. Am J Med. 2001;111:304-315.

167. Basran GS, Paul W, Morely J, Turner-Warwick M. Evidence in man of synergistic interaction between putative mediators of acute inflammation and asthma. Prostaglandins. 1982;1(8278):935-937.

168. Shibata M, Ohkubo T, Takahashi H, Inoki R. Interaction of bradykinin with substance P on vascular permeability and pain response. Japan J Pharmacol. 1986;41:427-429.

169. Noda M, Kariura Y, Pannasch U, et al. Neuroprotective role of bradykinin because of the attenuation of pro-inflammatory cytokine release from activated microglia. J Neurochem. 2007;101(2):397-410.

170. Ahn S-G, Jeong S-Y, Rhim H, Kim I-K. The role of c-Myc and heat shock protein 70 in human hepatocarcinoma Hep3B cells during apoptosis induced by prostaglandin A2/Δ12-prostaglandin J2. Biochim Biophys Acta. 1998;1448(1):115- 125.

171. Straus DS, Pascual G, Li M, et al. 15-deoxy-delta 12,14-prostaglandin J2 inhibits multiple steps in the NF-kappa B signaling pathway. Proc Natl Acad Sci U S A. 2000;97(9):4844-4849.

172. Peters HPF, Wiersma WC, Akkermans LM, et al. Gastrointestinal mucosal integrity after prolonged exercise with fluid supplementation. Med Sci Sports Exerc. 2000;32(1):134-142.

173. van Nieuwenhoven MA, Brouns F, Brummer RJ. Gastrointestinal profile of symptomatic athletes at rest and during physical exercise. Eur J Appl Physiol. 2004;91(4):429-434.

176

174. Pedersen BK, Toft AD. Effects of exercise on lymphocytes and cytokines. Br J Sports Med. 2000;34:246-251.

175. Suzuki K, Peake J, Nosaka K, et al. Changes in markers of muscle damage, inflammation and HSP70 after an Ironman Triathlon race. Eur J Appl Physiol. 2006;98(6):525-534.

176. Ostrowski K, Rohde T, Zacho M, Asp S, Pedersen BK. Evidence that interleukin-6 is produced in human skeletal muscle during prolonged running. J Physiol. 1998;508(3):949-953.

177. Cannon JG, Meydani SN, Fielding RA, et al. Acute phase response in exercise. II. Associations between vitamin E, cytokines, and muscle proteolysis. Am J Physiol Regul Integr Comp Physiol. 1991;260:1235-1240.

178. Fielding RA, Manfredi TJ, Ding W, Fiatarone MA, Evans WJ, Cannon J. Acute phase respone in exericse. III. Neutrophil and IL-1B accumulation in skeletal muscle. Am J Physiol Regul Integrative Comp Physiol. 1993;265:166-172.

179. de Oliveira EP, Burini RC. The impact of physical exercise on the gastrointestinal tract. Curr Opin Clin Nutr Metab Care. 2009;12(5):533-538.

180. Baumgart DC, Dignass AU. Intestinal barrier function. Curr Opin Clin Nutr Metab Care. 2002;5(6):685-694.

181. Lambert GP, Lang J, Bull A, et al. Fluid restriction during running increases GI permeability. Int J Sports Med. 2008;29(3):194-198.

182. Pals KL, Chang RT, Ryan AJ, Gisolfi CV. Effect of running intensity on intestinal permeability. J Appl Physiol. 1997;82(2):571-576.

183. Gathiram P, Gaffin SL, Brock-Utne JG, Wells MT. Prophylactic corticosteroid suppresses endotoxemia in heat-stressed primates. Aviat Space Environ Med. 1988;59(2):142-145.

184. Dokladny K, Moseley PL, Ma TY. Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability. Am J Physiol Gastrointest Liver Physiol. 2006;290(2):G204-212.

185. Bouchama A, Al-Sedairy S, Siddiqui S, Shail E, Rezeig M. Elevated pyrogenic cytokines in heatstroke. Chest. 1993;104:1498-1502.

186. Welc SS, Clanton TL, Dineen SM, Leon LR. Heat stroke activates a stress-induced cytokine response in skeletal muscle. J Appl Phys. 2013;115:1126-1137.

187. Chang DM. Role of cytokines in heat stroke. Immunol Invest. 1993;22(8):553-561.

177

188. Armstrong LE. Heat and Humidity. In: Bahrke MS, ed. Performance in Extreme Environments. Champaign, IL: Human Kinetics; 2000:15-70.

189. American College of Sports M, Armstrong LE, Casa DJ, et al. American College of Sports Medicine position stand. Exertional heat illness during training and competition. Med Sci Sports Exerc. 2007;39(3):556-572.

190. Casa DJ, Stearns RL, Lopez RM, et al. Influence of hydration on physiological function and performance during trail running in the heat. J Athl Train. 2010;45(2):147-156.

191. Boden BP, Breit I, Beachler JA, Williams A, Mueller FO. Fatalities in high school and college football players. Am J Sports Med. 2013;41(5):1108-1116.

192. Keren G, Epstein Y, Magazanik A. Temporary heat intolerance in a heatstroke patient. Aviat Space Envir Med. 1981;52(2):116-117.

193. Shibolet S, Coll R, Gilat T, Sohar E. Heatstroke: its clinical picture and mechanism in 36 cases. Q J Med. 1967;36(144):525-548.

194. Stringer v. Minnesota Vikings Football Club LLC. Supreme Court of Minnesota;2005.

195. Benzinger TH. On physical heat regulation and the sense of temperature in man. Proc Natl Acad Sci U S A. 1959;45:645-659.

196. Roberts WO. Exertional heat stroke during a cool weather marathon: a case study. Med Sci Sports Exerc. 2006;38(7):1197-1203.

197. Marshall JC. The gut as a potential trigger of exercise-induced inflammatory responses. Can J Physiol Pharmacol. 1998;76:479-484.

198. Bouchama A, Knochel JP. Heat stroke. N Engl J Med. 2002;346(25):1978-1988.

199. Gathiram P, Wells MT, Brock-Utne JG, Gaffin SL. Antilipopolysaccharide improves survival in primates subjected to heat stroke. Circ Shock. 1987;23(3):157-164.

200. Gathiram P, Wells MT, Brock-Utne JG, Wessels BC, Gaffin SL. Prevention of endotoxaemia by non-absorbable antibiotics in heat stress. J Clin Path. 1987;40:1364-1368.

201. OTC sales by category 2011-2014. Consumer Healthcare Products Association2015.

202. Treating pain with NSAID medications Consumer Reports2013.

203. Top 10 OTC brands by revenue in the U.S. in 2013. The Statistical Portal2013.

204. Warden SJ. Prophylactic use of NSAIDs by athletes: a risk/benefit assessment. Phys Sportsmed. 2010;1(38):1-7.

178

205. Feucht CL, Patel DR. Analgesics and anti-inflammatory medications in sports - use and abuse. Pediatr Clin N Am. 2010;57:751-774.

206. Tscholl P, Alonso JM, Dolle G, Junge A, Dvorak J. The use of drugs and nutritional supplements in top-level track and field athletes. Am J Sports Med. 2010;38(1):133- 140.

207. Suzic Lazic J, Dikic N, Radivojevic N, et al. Dietary supplements and medications in elite sport--polypharmacy or real need? Scand J Med Sci Sports. 2011;21(2):260-267.

208. Warner DC, Schnepf G, Barrett MS, Dian D, Swigonski NL. Prevalence, attitudes, and behaviors related to the use of nonsteroidal anti-inflammatory drugs (NSAIDs) in student athletes. J Adolescent Health. 2002;30:150-153.

209. Antman EM, Bennett JS, Daugherty A, Furberg C, Roberts H, Taubert KA. Use of nonsteroidal antiinflammatory drugs: an update for clinicians: a scientific statement from the American Heart Association. Circulation. 2007;115(12):1634-1642.

210. Lewis SC, Langman MJS, Laporte J-R, Matthews JNS, Rawlins MD, Wiholm B-E. Dose–response relationships between individual nonaspirin nonsteroidal anti- inflammatory drugs (NANSAIDs) and serious upper gastrointestinal bleeding: a meta-analysis based on individual patient data. Br J Clin Pharmacol. 2002;54:320- 326.

211. Wharam PC, Speedy DB, Noakes TD, Thompson JM, Reid SA, Holtzhausen LM. NSAID use increases the risk of developing hyponatremia during an Ironman triathlon. Med Sci Sports Exerc. 2006;38(4):618-622.

212. Nakahura T, Griswold W, Lemire J, Mendoza S, Rezni V. Nonsteroidal anti- inflammatory drug use in adolescence. J Adolescent Health. 1998;23:307-310.

213. Chambers CT, Reid GJ, McGrath PJ, Finley GA. Self-administration of over-the- counter medication for pain among adolescents. Arch Pediatr Adolesc Med. 1997;151:449-455.

214. Gilbertson RJ, Harris E, Pandey SK, Kelly P, Myers W. Paracetamol use, availability, and knowledge of toxicity among British and American adolescents. Arch Dis Child. 1996;75:194-198.

215. Huott MA, Storrow AB. A survey of adolescents’ knowledge regarding toxicity of over-the-counter medications. Acad Emerg Med. 1997;4:214-218.

216. Cook DB, O'Connor PJ, Eubanks SA, Smith JC, Lee M. Naturally occurring muscle pain during exercise: assessment and experimental evidence. Med Sci Sport Exerc. 1997;29(8):999-1012.

217. Gilbert JA. Acute and chronic effect of apirin on selected endurance variables. Sports Med Train Rehab. 1995;6(4):299-307.

179

218. Hudson GM, Green JM, Bishop PA, Richardson MT. Effects of caffeine and aspirin on light resistance training performance, perceived exertion, and pain perception. J Strength Cond Res. 2008;22(6):1950-1957.

219. Roi GS, Garagiola U, Verza P, et al. Aspirin does not affect exercise performance. Int J Sports Med. 1994;15(5):224-227.

220. Vane JR. Differential inhibition of cyclooxygenase isoforms an explanation of the action of NSAIDs. J Clin Rheumatol. 1998;4:S3-S10.

221. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin- like drugs. Nature. 1971;231(25):232-235.

222. Botting RM. Inhibitors of cyclooxygenases: mechanisms, selectivity and uses. J Physiol Pharmacol. 2006;57:113-124.

223. Capone ML, Tacconelli S, Di Francesco L, Sacchetti A, Sciulli MG, Patrignani P. Pharmacodynamic of cyclooxygenase inhibitors in humans. Prostaglandins Other Lipid Mediat. 2007;82:85-94.

224. Tacconelli S, Capone ML, Patrignani P. Clinical pharmacology of novel selective COX-2 inhibitors. Curr Pharm Des. 2004;10:589-601.

225. Knights KM, Mangoni AA, Miners JO. Defining the COX inhibitor selectivity of NSAIDs: implications for understanding toxicity. Expert Rev Clin Pharmacol. 2010;3(6):769-776.

226. Pairet M, van Ryn J. Experimental models used to investigate the differential inhibition of cyclooxygenase-1 and cyclooxygenase-2 by non-steroidal anti- inflammatory drugs. Inflamm Res. 1998;47(S2):S93-S101.

227. Ciccone CD. Davis's Drug Guide for Rehabilitation Specialists. Philadelphia, PA: F. A. Davis; 2013.

228. Ciccone CD. Pharmacology in Rehabiliation. 4 ed. Philadelphia, PA: F. A. Davis; 2007.

229. Patrono C, Garcia Rodriguez LA, Landolfi R, Baigent C. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med. 2005;353:2373-2383.

230. Patrignani P, Filabozzi P, Patrono C. Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects. J Clin Invest. 1982;69:1366-1372.

231. Cipollone F, Patrignani P, Greco A, et al. Differential suppression of thromboxane biosynthesis by indobufen and aspirin in patients with unstable angina. Circulation. 1997;96(4):1109-1116.

180

232. Pernerstorfer T, Schmid R, Bieglmayer C, Eichler H-G, Kapiotis S, Jilma B. Acetaminophen has greater antipyretic efficacy than aspirin in endotoxemia: a randomized, double-blind, placebo-controlled trial. Clin Pharmacol Ther. 1999;66:51-57.

233. Warner TD, Giuliano F, Vojnovic I, Bukasa A, Mitchell JA, Vane JR. Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis. Proc Natl Acad Sci U S A. 1999;96:7563-7568.

234. Sepehri G, Pourranjbar M, Moshtaghi-Kashanian G-R, Khachaki AS, Sepehri E. The effect of a prophylactic dose of ibuprofen on plasma level of interleukin 1, interleukin 6 and tumor necrosis factor-alpha in a 1500 m running practice. Am J Appl Sci. 2011;8(1):50-54.

235. Nieman DC, Henson DA, Dumke CL, et al. Ibuprofen use, endotoxemia, inflammation, and plasma cytokines during ultramarathon competition. Brain Behav Immun. 2006;20(6):578-584.

236. Michie HR, Manogue KR, Spriggs DR, et al. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med. 1988;318:1481-1486.

237. Spinas GA, Bloesch D, Keller U, Zimmerli W, Cammisuli S. Pretreatment with ibuprofen augments circulating tumor necrosis factor-a, interleukin-6, and elastase during acute endotoxinemia. J Infect Dis. 1991;163(1):89-95.

238. Martich GD, Danner RL, Ceska M, Suffredini AF. Detection of interleukin 8 and tumor necrosis factor in normal humans after intravenous endotoxin: the effects of antiinflammatory agents J Exp Med. 1991;173:1021-1024.

239. Collaboration AT. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. Br Med J. 2002;324:71-86.

240. Cryer B, Feldman M. Cyclooxygenase-1 and cyclooxygenase-2 selectivity of widely used nonsteroidal anti-inflammatory drugs. Am J Med. 1998;104:413-421.

241. Rhind SG, Gannon GA, Shephard RJ, Shek PN. Indomethacin modulates circulating cytokine responses to strenuous exercise in humans. Cytokine. 2002;19(3):153-158.

242. Rhind SG, Gannon GA, Suzui M, Shephard RJ, Shek PN. Indomethacin inhibits circulating PGE2 and reverses postexercise suppression of natural killer cell activity. Am J Physiol Regul Integr Comp Physiol. 1999;276:R1496-R1505.

243. Masso Gonzalez EL, Patrignani P, Tacconelli S, Garcia Rodriguez LA. Variability among nonsteroidal antiinflammatory drugs in risk of upper gastrointestinal bleeding. Arthritis Rheum. 2010;62(6):1592-1601.

181

244. Todd PA, Clissold SP. Naproxen. A reappraisal of its pharmacology, and therapeutic use in rheumatic diseases and pain states. Drugs. 1990;40(1):91-137.

245. Capone ML, Tacconelli S, Sciulli MG, et al. Clinical pharmacology of platelet, monocyte, and vascular cyclooxygenase inhibition by naproxen and low-dose aspirin in healthy subjects. Circulation. 2004;109(12):1468-1471.

246. Garcia Rodriguez LA, Gonzalez-Perez A. Long-term use of non-steroidal anti- inflammatory drugs and the risk of myocardial infarction in the general population. BMC Med. 2005;3:17-22.

247. Capone ML, Tacconelli S, Sciulli MG, et al. Human pharmacology of naproxen sodium. J Pharmacol Exp Ther. 2007;322(2):453-460.

248. Vane JR, Botting RM. Anti-inflammatory drugs and their mechanism of action. Inflamm Res. 1998;47:S78-S87.

249. Bombardier CB, Laine L, Reicin A, et al. Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. N Engl J Med. 2000;343:1520-1528.

250. Shah AA, Thjodleifsson B, Murray FE, et al. Selective inhibition of COX-2 in humans is associated with less gastrointestinal injury: a comparison of nimesulide and naproxen. Gut. 2001;48:339-346.

251. Smecuol E, Bai JC, Sugai E, et al. Acute gastrointestinal permeability responses to different non-steroidal anti-inflammatory drugs. Gut. 2001;49:6.

252. Gibson GR, Whitacre EB, Ricotti CA. Colitis induced by nonsteroidal anti- inflammatory drugs. Arch Intern Med. 1992;152:625-632.

253. Crawshaw LI, Wallace H, Crabbe J. Ethanol, body temperature, and thermoregulation Clin Exp Pharmacol Physiol. 1998;25:6.

254. Saverymuttu SH, Thomas A, Grundy A, Maxwell JD. Ileal stricturing after long term indomethacin treatment. Postgrad Med J. 1986;62:267-268.

255. Laine L, Curtis SP, Langman M, et al. Lower gastrointestinal events in a double-blind trial of the cyclo-oxygenase-2 selective inhibitor etoricoxib and the traditional nonsteroidal anti-inflammatory drug diclofenac. Gastroenterology. 2008;135(5):1517-1525.

256. Laine L, Connors LG, Reicin A, et al. Serious lower gastrointestinal clinical events with nonselective NSAID or coxib use. Gastroenterology. 2003;124(2):288-292.

257. Mokhtare M, Valizadeh SM, Emadian O. Lower gastrointestinal bleeding due to nonsteroid anti-inflammatory drug-induced colopathy case report and literature review. Middle East J Dig Dis. 2013;5:107-111.

182

258. Appleyard CB, McCafferty D-M, Tigley AW, Swain MG, Wallace JL. Tumor necrosis factor mediatio nof NSAID-induced gastric damage: role of leukocyte adherence. Am J Physiol Gastrointest Liver Physiol. 1996;270:G42-G48.

259. Bjarnason I, Williams P, Smethurst P, Peters TJ, Levi AJ. Effect of non-steroidal anti-inflammatory drugs and prostaglandins on the permeability of the human small intestine. Gut. 1986;27:6.

260. Lambert GP, Schmidt A, Schwarzkopf K, Lanspa S. Effect of aspirin dose on gastrointestinal permeability. Int J Sports Med. 2012;33(6):421-425.

261. Davies GR, Wilkie ME, Rampton DS. Effects of metronidazole and misoprostol on indomethacin-induced changes in intestinal permeability. Dig Dis Sci. 1993;38(3):417-425.

262. Bjarnason I, Smethurst P, Fenn CG, Lee CE, Menzies I, Levi AJ. Misoprostol reduces indomethacin-induced changes in human small intestinal permeability. Dig Dis Sci. 1989;34(3):407-411.

263. Bjarnason I, Takeuchi K. Intestinal permeability in the pathogenesis of NSAID- induced enteropathy. J Gastroenterol. 2009;44 23-29.

264. Lichtenberger LM, Zhou Y, Jayaraman V, et al. Insight into NSAID-induced membrane alterations, pathogenesis and therapeutics: characterization of interaction of NSAIDs with phosphatidylcholine. Biochim Biophys Acta. 2012;1821(7):994- 1002.

265. Giraud MN, Motta C, Romero JJ, Bommelauer G, Lichtenberger LM. Interaction of indomethacin and naproxen with gastric surface-active phospholipids: a possible mechanism for the gastric toxicity of nonsteroidal anti-inflammatory drugs (NSAIDs). Biochem Pharmcol. 1999;57:247-254.

266. Bjarnason I, Smethurst P, MacPherson A, et al. Glucose and citrate reduce the permeability changes caused by indomethacin in humans. Gastroenterol. 1992;102(5):1546-1550.

267. Haag MDM, Bos MJ, Hofman A, Koudstaal PJ, Breteler MMB, Stricker BHC. Cyclooxygenase selectivity of nonsteroidal anti-inflammatory drugs and risk of stroke. Arch Intern Med. 2008;168(11):1219-1224.

268. Schafer AI. Effects of nonsteroidal antiinflammatory drugs on platelet function and systemic hemostasis. J Clin Pharmacol. 1995;35:209-219.

269. Bruning RS, Dahmus JD, Kenney WL, Alexander LM. Aspirin and clopidogrel alter core temperature and skin blood flow during heat stress. Med Sci Sports Exerc. 2013;45(4):674-682.

183

270. Kim S, Wook Joo K. Electrolyte and acid-base disturbances associated with nonsteroidal anti-inflammatory drugs. Electrolyte Blood Press. 2007;5:116-125.

271. Cheng HF, Harris RC. Renal effects of non-steroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors. Curr Pharm Des. 2005;11(14):1795-1804.

272. Rosner MH, Kirven J. Exercise-associated hyponatremia. Clin J Am Soc Nephrol. 2007;2(1):151-161.

273. Davis DP, Videen JS, Marino A, et al. Exercise-associated hyponatremia in marathon runners: a two year experience. J Emerg Med. 2001;21(1):47-57.

274. Hsieh M, Roth R, Davis DL, Larrabee H, Callaway CW. Hyponatremia in runners requiring on-site medical treatment at a single marathon. Med Sci Sport Exerc. 2002;34(2):185-189.

275. Almond CS, Shin AY, Fortescue EB, et al. Hyponatremia among runners in the Boston Marathon. N Engl J Med. 2005;352(15):1550-1556.

276. Jacobson ED, Bass DE. Effects of sodium salicylate on physiological responses to work in heat. J Appl Phys. 1964;19(1):33-36.

277. Downey JA, Darling RC. Effect of salicylates on elevation of body temperature during exercise. J Appl Phys. 1962;17(2):323-325.

278. Bradford CD, Cotter JD, Thorburn MS, Walker RJ, Gerrard DF. Exercise can be pyrogenic in humans. Am J Physiol Regul Integr Comp Physiol. 2007;292(1):R143- 149.

279. Starkie RL, Hargreaves M, Rolland J, Febbraio MA. Heat stress, cytokines, and the immune response to exercise. Brain Behav Immun. 2005;19(5):404-412.

280. Pescatello LS, ed ACSM's Guidelines for Exercise Testing and Prescription. 9th ed. Baltimore, MD: Lippincott Williams & Wilkins; 2014. Arena R, ed. Exercise Testing.

281. PAR-Q & YOU. In: Physiology CSfE, ed2002.

282. Preparticipation Physical Examination Form. In: Medicine AMSfS, ed2010.

283. McAnulty SR, Owens JT, McAnulty LS, et al. Ibuprofen use during extreme exercise: effects on oxidative stress and PGE2. Med Sci Sports Exerc. 2007;39(7):1075-1079.

284. Northridge DB, Grant S, Ford I, et al. Novel exercise protocol suitable for use on a treadmill or a bicycle ergometer. Br Heart J. 1990;64:313-316.

184

285. Lam NYL, Rainer TH, Chiu RWK, Lo YMD. EDTA is a better anticoagulant than heparin or citrate for delayed blood processing for plasma DNA analysis. Clin Chem. 2004;50(1):256-257.

286. van Wijck K, Verlinden TJ, van Eijk HM, et al. Novel multi-sugar assay for site- specific gastrointestinal permeability analysis: a randomized controlled crossover trial. Clin Nutr. 2013;32(2):245-251.

287. Farhadi A, Keshavarzian A, Holmes EW, Fields J, Zhang L, Banan A. Gas chromatographic method for detection of urinary sucralose: application to the assessment of intestinal permeability. J Chromatogr. 2003;784:145-154.

288. American College of Sports M, Sawka MN, Burke LM, et al. American College of Sports Medicine position stand. Exercise and fluid replacement. Med Sci Sports Exerc. 2007;39(2):377-390.

289. Popowski RA, Oppliger RA, Lambert GP, Johnson RF, Johnson AK, Gisolfi CV. Blood and urinary measures of hydration status during progressive acute dehydration. Med Sci Sport Exerc. 2001;33(5):747-753.

290. Atago. Clinical refractometers. 2007.

291. Sawka MN, Wenger CB. Physiological responses to acute exercise-heat stress. Natick, MA: U.S. Army Research Institute of Environmental Medicine; 1988.

292. Casa DJ, Becker SM, Ganio MS, et al. Validity of devices that assess body temperature during outdoor exercise in the heat. J Athl Train. 2007;42(3):333-342.

293. Ganio MS, Brown CM, Casa DJ, et al. Validity and reliability of devices that assess body temperature during indoor exercise in the heat. J Athl Train. 2009;44(2):124- 135.

294. Pfeiffer B, Stellingwerff T, Hodgson AB, et al. Nutritional intake and gastrointestinal problems during competitive endurance events. Med Sci Sports Exerc. 2012;44(2):344-351.

295. Lambert GP, Murray R, Eddy D, Scott W, Laird R, Gisolfi CV. Intestinal permeability following the 1998 Ironman Triathlon. Med Sci Sport Exerc. 1999;31(5):S318.

185

Cycling, no thermal stress

NSAIDs and thermal stress

+ P[Na ] HR and BP TNF-α and IL-6 GI distress and Posm permeability

Plasma LPS

Significant Tc

Performance

Figure 5.1. Working Model for Overall Study. NSAIDs will exacerbate responses seen during exercise and no thermal stress, increasing Tc through immune, GI, and cardiovascular pathways.

186

3 Days – 1 Day Prior to Data Data Collection (4 Trials) Information Collection

Informed Consent Begin diet and Arrive to Lab Cycling Trial Immediately 3hrs Post- Form physical activity Post-Exercise Explain study log 3 days prior Confirm 2 pills Continuous Tc 3.5 ml/kg water procedures Report to lab 1 taken and HR Blood every 15 min rd Health and Injury day prior to pick Give 3 pill BP, RPE, and GI Urine Tc every 5 min History up Void bladder, symptoms every BM Post Questionnaire Take Home ingest sugar 15min Tc Blood STEEP test Instructions solution 3.5 kg/mL water BP, HR, RPE Urine Random group 3 pills Baseline every 15 min GI symptoms BM, BP, HR assignment Fecal occult Blood Blood at 80 min GI symptoms Take Home Urine 187 Tc

HR, BP GI symptoms

Figure 5.2. Experimental Procedures Schematic. Includes information session and data collection. Blood measures include plasma osmolality, plasma sodium concentration, and cytokines TNF-α and IL-6. Urine measures include sugar excretion and urine specific gravity. Abbreviations: BM = body mass; BP = blood pressure; HR = heart rate; GI = gastrointestinal; RPE = rate of perceived exertion; Tc = core temperature.

BIBLIOGRAPHY

Ahn S-G, Jeong S-Y, Rhim H, Kim I-K. The role of c-Myc and heat shock protein 70 in human hepatocarcinoma Hep3B cells during apoptosis induced by prostaglandin A2/Δ12- prostaglandin J2. Biochim Biophys Acta. 1998;1448(1):115-125.

Akira S, Hirano T, Taga T, Kishimoto T. Biology of multifunctional cytokines: IL 6 and related molecules (IL 1 and TNF). FASEB J. 1990;4:2860-2867.

Almond CS, Shin AY, Fortescue EB, et al. Hyponatremia among runners in the Boston Marathon. N Engl J Med. 2005;352(15):1550-1556.

American College of Sports M, Armstrong LE, Casa DJ, et al. American College of Sports Medicine position stand. Exertional heat illness during training and competition. Med Sci Sports Exerc. 2007;39(3):556-572.

American College of Sports M, Sawka MN, Burke LM, et al. American College of Sports Medicine position stand. Exercise and fluid replacement. Med Sci Sports Exerc. 2007;39(2):377-390.

Antman EM, Bennett JS, Daugherty A, Furberg C, Roberts H, Taubert KA. Use of nonsteroidal antiinflammatory drugs: an update for clinicians: a scientific statement from the American Heart Association. Circulation. 2007;115(12):1634-1642.

Appleyard CB, McCafferty D-M, Tigley AW, Swain MG, Wallace JL. Tumor necrosis factor mediation of NSAID-induced gastric damage: role of leukocyte adherence. Am J Physiol Gastrointest Liver Physiol. 1996;270:G42-G48.

Armstrong LE, Johnson EC, Casa DJ, et al. The American football uniform: uncompensable heat stress and hyperthermic exhaustion. J Athl Train. 2010;45(2):11.

Armstrong LE, Pumerantz AC, Fiala KA, et al. Human hydration indices: acute and longitudinal reference values. Int J Sport Nutr Exerc Metab. 2010;20(2):145-153.

Armstrong LE. Heat and Humidity. In: Bahrke MS, ed. Performance in Extreme Environments. Champaign, IL: Human Kinetics; 2000:15-70.

Aronoff DM, Neilson EG. Antipyretics: mechanisms of action and clinical use in fever suppression. Am J Med. 2001;111:304-315.

Ashton T, Young I, Davison G, et al. Exercise-induced endotoxemia: the effect of ascorbic acid supplementation. Free Radical Bio Med. 2003;35(3):284-291.

188

Atago. Clinical refractometers. 2007.

Baert FJ, R. DHG, Peeters M, et al. Tumour necrosis factor a antibody (infliximab) therapy profoundly downregulates the inflammation in Chrohn's ileocolitis. Gastroenterol. 1999;116:22-28.

Bandyopadhyay B, Bandyopadhyay SK. Role of prostaglandin in the regulation of gastric H+ transporting system. Indian J Clin Biochem. 1998;13(1):41-45.

Basran GS, Paul W, Morely J, Turner-Warwick M. Evidence in man of synergistic interaction between putative mediators of acute inflammation and asthma. Prostaglandins. 1982;1(8278):935-937.

Baumgart DC, Dignass AU. Intestinal barrier function. Curr Opin Clin Nutr Metab Care. 2002;5(6):685-694.

Befrits R, Johannsen C. Oral PGE2 inhibits gastric acid secretion in man. Prostaglandins. 1985;29(1):143-152.

Benzinger TH. On physical heat regulation and the sense of temperature in man. Proc Natl Acad Sci U S A. 1959;45:645-659.

Berg A, Muller H-M, Rathmann S, Deibert P. The gastrointestinal system - an essential target organ of the athlete's health and physical performance. Exerc Immunol Rev. 1999;5:78-95.

Berglund JJ, Riegler M, Zolotarevsky Y, Wenzl E, Turner JR. Regulation of human jejunal transmucosal resistance and MLC phosphorylation by Na+-glucose cotransport. Am J Physiol Gastrointest Liver Physiol. 2001;281:G1487-G1493.

Berkes J, Viswanathan VK, Savkovic SD, Hecht G. Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation. Gut. 2003;52:439-451.

Berne RM, Levy MN. Principles of Physiology. 3rd ed. St. Louis, MO: Mosby, Inc; 2000.

Beutler B, Cerami A. The biology of cachectin/TNF - a primary mediator of the host response. Ann Rev Immunol. 1989;7:625-655.

Binkley HM, Beckett J, Casa DJ, Kleiner DM, Plummer PE. National Athletic Trainers' Association position statement: exertional heat illnesses. J Athl Train. 2002;37(3):329-343.

Bjarnason I, Peters TJ, Levi AJ. Intestinal permeability: clinical correlates. Dig Dis. 1986;4(2):83-92.

Bjarnason I, Smethurst P, Fenn CG, Lee CE, Menzies I, Levi AJ. Misoprostol reduces indomethacin-induced changes in human small intestinal permeability. Dig Dis Sci. 1989;34(3):407-411.

189

Bjarnason I, Smethurst P, MacPherson A, et al. Glucose and citrate reduce the permeability changes caused by indomethacin in humans. Gastroenterol. 1992;102(5):1546-1550.

Bjarnason I, Takeuchi K. Intestinal permeability in the pathogenesis of NSAID-induced enteropathy. J Gastroenterol. 2009;44 23-29.

Bjarnason I, Williams P, Smethurst P, Peters TJ, Levi AJ. Effect of non-steroidal anti- inflammatory drugs and prostaglandins on the permeability of the human small intestine. Gut. 1986;27:6.

Blevins R, Apel T. Doctor: Wilbanks' death unpreventable, freak occurrence. The Clarion- Ledger2014.

Blikslager AT, Roberts MC, Rhoads JM, Argenzio RA. Prostaglandins I2 and E2 have a synergistic role in rescuing epithelial barrier function in porcine ileum. J Clin Invest. 1997;100(8):1928-1933.

Boden BP, Breit I, Beachler JA, Williams A, Mueller FO. Fatalities in high school and college football players. Am J Sports Med. 2013;41(5):1108-1116.

Bombardier CB, Laine L, Reicin A, et al. Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. N Engl J Med. 2000;343:1520-1528.

Boone M, Deen PM. Physiology and pathophysiology of the vasopressin-regulated renal water reabsorption. Pflugers Arch. 2008;456(6):1005-1024.

Botting RM. Inhibitors of cyclooxygenases: mechanisms, selectivity and uses. J Physiol Pharmacol. 2006;57:113-24.

Bouchama A, Al-Sedairy S, Siddiqui S, Shail E, Rezeig M. Elevated pyrogenic cytokines in heatstroke. Chest. 1993;104:1498-1502.

Bouchama A, Knochel JP. Heat stroke. N Engl J Med. 2002;346(25):1978-1988.

Bouchama A, Parhar RS, El-Yazigi A, Sheth K, Al-Sedairy S. Endotoxemia and release of tumor necrosis factor and interleukin Ia in acute heatstroke. J Appl Physiol. 1991;70(6):2640-2644.

Bradford CD, Cotter JD, Thorburn MS, Walker RJ, Gerrard DF. Exercise can be pyrogenic in humans. Am J Physiol Regul Integr Comp Physiol. 2007;292(1):R143-149.

Brock TG, McNish RW, Peters-Golden M. Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2. J Biol Chem. 1999;274(17):11660-11666.

Brocker C, Thompson D, Matsumoto A, Nebert DW, Vasiliou V. Evolutionary divergence and functions of the human interleukin (IL) gene family. Hum Genom. 2010;5(1):30-55.

190

Brock-Utne JG, Gaffin SL, Wells MT, et al. Endotoxaemia in exhausted runners after a long-distance race. S Afr Med J. 1988;73(9):533-536.

Brooks M. Ten years after Korey Stringer's death, heat-related deaths still plague football teams around the country. The Washington Post. August 12, 2011, 2011.

Bruggeman TM, Wood JG, Davenport HW. Local control of blood flow in the dog's stomach: vasodilation caused by acid back diffusion following topical application of salicylic acid. Gastroenterol. 1979;77:736-744.

Bruning RS, Dahmus JD, Kenney WL, Alexander LM. Aspirin and clopidogrel alter core temperature and skin blood flow during heat stress. Med Sci Sports Exerc. 2013;45(4):674- 682.

Calabresi L. High-density lipoproteins protect isolated rat hearts from ischemia- reperfusion injury by reducing cardiac tumor necrosis factor-alpha content and enhancing prostaglandin release. Cir Res. 2002;92(3):330-337.

Camus G, Nys M, Poortmans JR, et al. Endotoxemia, production of tumor necrosis factor- a and polymorphonuclear neutrophil activation following strenous exercise in humans. Eur J Appl Physiol. 1998;79:62-68.

Cannon JG, Meydani SN, Fielding RA, et al. Acute phase response in exercise. II. Associations between vitamin E, cytokines, and muscle proteolysis. Am J Physiol Regul Integr Comp Physiol. 1991;260:1235-1240.

Canny G, Levy O. Bactericidal/permeability-increasing protein (BPI) and BPI homologs at mucosal sites. Trends Immunol. 2008;29(11):541-547.

Capone ML, Tacconelli S, Di Francesco L, Sacchetti A, Sciulli MG, Patrignani P. Pharmacodynamic of cyclooxygenase inhibitors in humans. Prostaglandins Other Lipid Mediat. 2007;82:85-94.

Capone ML, Tacconelli S, Sciulli MG, et al. Clinical pharmacology of platelet, monocyte, and vascular cyclooxygenase inhibition by naproxen and low-dose aspirin in healthy subjects. Circulation. 2004;109(12):1468-1471.

Capone ML, Tacconelli S, Sciulli MG, et al. Human pharmacology of naproxen sodium. J Pharmacol Exp Ther. 2007;322(2):453-60.

Carden DL, Granger DN. Pathophysiology of ischemia-reperfusion injury. J Pathol. 2000;190:255-266.

Casa DJ, Armstrong LE, Hillman SK, et al. National Athletic Trainers' Association Position Statement: fluid replacement for athletes. J Athl Train. 2000;35(2):212-224.

Casa DJ, Becker SM, Ganio MS, et al. Validity of devices that assess body temperature during outdoor exercise in the heat. J Athl Train. 2007;42(3):333-42.

191

Casa DJ, DeMartini JK, Bergeron MF, et al. National Athletic Trainers' Association position statement: exertional heat illnesses. J Athl Train. 2015;50(9):986-1000.

Casa DJ, Stearns RL, Lopez RM, et al. Influence of hydration on physiological function and performance during trail running in the heat. J Athl Train. 2010;45(2):147-156.

Casa DJ. Exercise in the heat. II. Critical concepts in rehydration, exertional heat illnesses, and maximizing athletic performance. J Athl Train. 1999;34(3):253-262.

Chambers CT, Reid GJ, McGrath PJ, Finley GA. Self-administration of over-the-counter medication for pain among adolescents. Arch Pediatr Adolesc Med. 1997;151:449-455.

Chang DM. Role of cytokines in heat stroke. Immunol Invest. 1993;22(8):553-561.

Chen L, Yang G, Grosser T. Prostanoids and inflammatory pain. Prostaglandins Other Lipid Mediat. 2013;104-105:58-66.

Cheng HF, Harris RC. Renal effects of non-steroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors. Curr Pharm Des. 2005;11(14):1795-1804.

Christou DD, Jones PP, Jordan J, Diedrich A, Robertson D, Seals DR. Women have lower tonic autonomic support of arterial blood pressure and less effective baroreflex buffering than men. Circulation. 2005;111(4):494-498.

Ciccone CD. Davis's Drug Guide for Rehabilitation Specialists. Philadelphia, PA: F. A. Davis; 2013.

Ciccone CD. Pharmacology in Rehabiliation. 4 ed. Philadelphia, PA: F. A. Davis; 2007.

Cipollone F, Patrignani P, Greco A, et al. Differential suppression of thromboxane biosynthesis by indobufen and aspirin in patients with unstable angina. Circulation. 1997;96(4):1109-1116.

Clausen T. Hormonal and pharmacological modification of plasma potassium homeostasis. Fundam Clin Pharmacol. 2010;24(5):595-605.

Clements JM, Casa DJ, Knight JC, et al. Ice-water immersion and cold-water immersion provide similar cooling rates in runners with exercise-induced hyperthermia. J Athl Train. 2002;37(2):146-150.

Collaboration AT. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. Br Med J. 2002;324:71-86.

Collard CD, Gelman S. Pathophysiology, clinical manifestations, and prevention of ischemia–reperfusion injury. Anesthesiology. 2001;94:1133-1138.

192

Collings KL, Pierce Pratt F, Rodriguez-Stanley S, Bemben M, Miner PB. Esophageal reflux in conditioned runners, cyclists, and weightlifters. Med Sci Sports Exerc. 2003;35(5):730-735.

Commins SP, Borish L, Steinke JW. Immunologic messenger molecules: cytokines, interferons, and chemokines. J Allergy Clin Immunol. 2010;125(2 Suppl 2):S53-S72.

Constanti A, Bartke A, Khardori R. Basic Endocrinology. Amsterdam, Netherlands: Harwood Academic Publishers; 1998.

Cook DB, O'Connor PJ, Eubanks SA, Smith JC, Lee M. Naturally occurring muscle pain during exercise: assessment and experimental evidence. Med Sci Sport Exerc. 1997;29(8):999-1012.

Cook DB, O'Connor PJ, Oliver SE, Lee Y. Sex differences in naturally occurring leg muscle pain and exertion during maximal cycle ergometry. Intern J Neuroscience. 1998;95:183-202.

Crawshaw LI, Wallace H, Crabbe J. Ethanol, body temperature, and thermoregulation. Clin Exp Pharmacol Physiol. 1998;25:6.

Creamer M, Hagedorn E. Family in disbelief that drinking too much water killed football player. The Bakersfield Californian2008.

Cryer B, Feldman M. Cyclooxygenase-1 and cyclooxygenase-2 selectivity of widely used nonsteroidal anti-inflammatory drugs. Am J Med. 1998;104:413-421.

Da Silva E, Pinto RS, Cadore EL, Kruel LF. Nonsteroidal anti-inflammatory drug use and endurance during running in male long-distance runners. J Athl Train. 2015;50(3):295-302.

Darling RL, Romero JJ, Dial EJ, Akunda JK, Langenbach R, Lichtenberger LM. The effects of aspirin on gastric mucosal integrity, surface hydrophobicity, and prostaglandin metabolism in cyclooxygenase knockout mice. Gastroenterology. 2004;127(1):94-104.

Davies GR, Wilkie ME, Rampton DS. Effects of metronidazole and misoprostol on indomethacin-induced changes in intestinal permeability. Dig Dis Sci. 1993;38(3):417- 425.

Davies NM. Review article: non-steroidal anti-inflammatory drug-induced gastrointestinal permeability. Aliment Pharmacol Ther. 1998;12:303-320.

Davis DP, Videen JS, Marino A, et al. Exercise-associated hyponatremia in marathon runners: a two year experience. J Emerg Med. 2001;21(1):47-57.

de Oliveira EP, Burini RC. The impact of physical exercise on the gastrointestinal tract. Curr Opin Clin Nutr Metab Care. 2009;12(5):533-538.

193

Del Prete G, De Carli M, Almerigogna F, Grazia Giudizi M, Biagiotti R, Romagnani S. Human IL-10 is produced by both type 1 helper (Thl) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production. 1993;150(2):353- 360.

Ding L, Linsley PS, Haung LY, Germain RN, Shevach EM. IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression. J Immunol. 1993;151(3):1224-1234.

Dokladny K, Moseley PL, Ma TY. Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability. Am J Physiol Gastrointest Liver Physiol. 2006;290(2):G204-212.

Domschke W, Domschke S, Hornig D, Demling L. Prostaglandin-stimulated gastric mucus secretion in man. Acta Hepto-Gastroenterol. 1978;25:292-294.

Doran JE. Biological effects of endotoxin. In: Kraft CH, ed. Gut-derived infectious toxic shock: current studies in hematology and blood transfusion. Vol 59: Basel Krager; 1992:66-99.

Downey JA, Darling RC. Effect of salicylates on elevation of body temperature during exercise. J Appl Phys. 1962;17(2):323-325.

Dumke CL, Nieman DC, Oley K, Lind RH. Ibuprofen does not affect serum electrolyte concentrations after an ultradistance run. Br J Sports Med. 2007;41(8):492-496.

Dunn MJ, Greely HP, Valtin H, Kintner LB, Beeuwkes R. Renal excretion of prostaglandins E2 and F2alpha in diabetes insipidus rats. Am J Physiol. 1978;235(6):E624- E627.

Dunn MJ, Zambraski EJ. Renal effects of drugs that inhibit prostagiandin synthesis. Kidney Int. 1980;18:609-622.

Dusting GJ, Moncada S, Vane JR. Disappearance of prostacyclin (PGI2) in the circulation of the dog [proceedings]. Br J Pharmacol. 1978;62(3):414P-415P.

Ensor JE, Wiener SM, McCrea KA, Viscardi RM, Crawford EK, Hasday JD. Differential effects of hyperthermia on macrophage interleukin-6 and tumor necrosis factor-alpha expression. Am J Physiol Cell Physiol. 1994;266:C967-C974.

Epstein Y, Shapiro Y, Brill S. Role of surface area-to-mass ratio and work efficiency in heat intolerance. J Appl Physiol. 1983;54(3):831-836.

Erickson RA, Tarnawski A, Dines G, Stachura J. 16,16-Dimethyl prostaglandin E2 induces villus contraction in rats without affecting intestinal restitution. Gastroenterol. 1990;99(3).

Facciabene A, Motz GT, Coukos G. T-regulatory cells: key players in tumor immune escape and angiogenesis. Cancer Res. 2012;72(9):2162-2171.

194

Farhadi A, Keshavarzian A, Holmes EW, Fields J, Zhang L, Banan A. Gas chromatographic method for detection of urinary sucralose: application to the assessment of intestinal permeability. J Chromatogr. 2003;784:145-154.

Faul F, Erdfelder E, Lang A-G, Buchner A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods. 2007;39(2):175-191.

Ferraz J-GP, McKnight W, Sharkey KA, Wallace JL. Impaired vasodilatory responses in the gastric microcirculation of anesthetized rats with secondary biliary cirrhosis. Gastroenterol. 1995;108:1183-1191.

Feucht CL, Patel DR. Analgesics and anti-inflammatory medications in sports - use and abuse. Pediatr Clin N Am. 2010;57:751-74.

Fielding RA, Manfredi TJ, Ding W, Fiatarone MA, Evans WJ, Cannon J. Acute phase respone in exericse. III. Neutrophil and IL-1B accumulation in skeletal muscle. Am J Physiol Regul Integrative Comp Physiol. 1993;265:166-172.

Flores D, Liu Y, Liu W, Satlin LM, Rohatgi R. Flow-induced prostaglandin E2 release regulates Na and K transport in the collecting duct. Am J Physiol Renal Physiol. 2012;303:F632-F638.

Foster J, Taylor L, Chrismas BC, Watkins SL, Mauger AR. The influence of acetaminophen on repeated sprint cycling performance. Eur J Appl Physiol. 2014;114(1):41-8.

Franscesconi RP, Willis JS, Gaffin SL, Hubbard RW. On the trail of potassium in heat injury. Wilderness Environ Med. 1997;8:105-110.

Gagnon D, Kenny GP. Does sex have an independent effect on thermoeffector responses during exercise in the heat? J Physiol. 2012;590(23):5963-73.

Ganio MS, Brown CM, Casa DJ, et al. Validity and reliability of devices that assess body temperature during indoor exercise in the heat. J Athl Train. 2009;44(2):124-135.

Garcia Rodriguez LA, Gonzalez-Perez A. Long-term use of non-steroidal anti- inflammatory drugs and the risk of myocardial infarction in the general population. BMC Med. 2005;3:17-22.

Garella S, Matarese RA. Renal effects of prostaglandins and clinical adverse effects of nonsteroidal anti-inflammatory agents. Medicine. 1984;63(3):165-181.

Gathiram P, Gaffin SL, Brock-Utne JG, Wells MT. Prophylactic corticosteroid suppresses endotoxemia in heat-stressed primates. Aviat Space Environ Med. 1988;59(2):142-145.

Gathiram P, Wells MT, Brock-Utne JG, Gaffin SL. Antilipopolysaccharide improves survival in primates subjected to heat stroke. Circ Shock. 1987;23(3):157-164.

195

Gathiram P, Wells MT, Brock-Utne JG, Wessels BC, Gaffin SL. Prevention of endotoxaemia by non-absorbable antibiotics in heat stress. J Clin Path. 1987;40:1364- 1368.

Gazzano-Santoro H, Parent JB, Grinna L, et al. High-affinity binding of the bactericidal/permeability-increasing protein and a recombinant amino-termial fragment to the lipid A region of lipopolysaccharide. Infect Immun. 1992;60(11):4754-5761.

Gibson GR, Whitacre EB, Ricotti CA. Colitis induced by nonsteroidal anti-inflammatory drugs. Arch Intern Med. 1992;152:625-632.

Gilbert JA. Acute and chronic effect of apirin on selected endurance variables. Sports Med Train Rehab. 1995;6(4):299-307.

Gilbertson RJ, Harris E, Pandey SK, Kelly P, Myers W. Paracetamol use, availability, and knowledge of toxicity among British and American adolescents. Arch Dis Child. 1996;75:194-198.

Gill SK, Allerton DM, Ansley-Robson P, Hemmings K, Cox M, Costa RJ. Does short-term high dose probiotic supplementation containing lactobacillus casei attenuate exertional- heat stress induced endotoxaemia and cytokinaemia? Int J Sport Nutr Exerc Metab. 2015.

Gill SK, Teixeira A, Rama L, et al. Circulatory endotoxin concentration and cytokine profile in response to exertional-heat stress during a multi-stage ultra-marathon competition. Exerc Immunol Rev. 2015;21:114-28.

Giraud MN, Motta C, Romero JJ, Bommelauer G, Lichtenberger LM. Interaction of indomethacin and naproxen with gastric surface-active phospholipids: a possible mechanism for the gastric toxicity of nonsteroidal anti-inflammatory drugs (NSAIDs). Biochem Pharmcol. 1999;57:247-254.

Goodman CC, Fuller KS. Pathology: Implications for the Physical Therapist. 3rd ed. St. Louis, MO: Saunders Elsevier; 2009.

Gookin JL, Galanko JA, Blikslager AT, Argenzio RA. PG-mediated closure of paracellular pathway and not restitution is the primary determinant of barrier recovery in acutely injured porcine ileum. Am J Physiol Gastrointest Liver Physiol. 2003;285:G967-G979.

Graham DY, Agrawal NM, Campbell DR, et al. Ulcer prevention in long-term users of nonsteroidal anti-inflammatory drugs. Results of a double-blind, randomized, multicenter, active- and placebo-controlled study of misoprostol vs lansoprazole. Arch Intern Med. 2002;162:169-175.

Granger DN, Richardson PDI, Kvietys PR, Mortillaro NA. Intestinal blood flow. Gastroenterol. 1980;78(4):837-863.

196

Grazia Roncarolo M, Gregori S, Battaglia M, Bacchetta R, Fleischhauer K, Levings MK. Interleukin-10-secreting type 1 regulatory T cells in rodents and humans. Immunol Rev. 2006;212:28-50.

Gretzer B, Maricic N, Respondek M, Schuligoi R, Peskar B. Effects of specific inhibition of cyclo-oxygenase-1 and cyclo-oxygenase-2 in the rat stomach with normal mucosa and after acid challenge. Br J Pharmacol. 2001;132:1565-1573.

Grisham MB, Hernandez LA, Granger DN. Xanthine oxidase and neutrophil infiltration in intestinal ischemia. Am J Physiol Gastrointest Liver Physiol. 1986;251:G567-G574.

Grossman WJ, Verbsky JW, Barchet W, Colonna M, Atkinson JP, Ley TJ. Human T regulatory cells can use the perforin pathway to cause autologous target cell death. Immunity. 2004;21(4):589-601.

Gryglewski RJ, Szczeklik A, Wandzilak M. The effect of six prostaglandins, prostacyclin and iloprost on generation of superoxide anions by human polymorphonuclear leukocytes stimulated by zymosan or formyl-methionyl-leucyl-phenylalanine. Biochem Pharmcol. 1987;36(24):4209-4213.

Haag MDM, Bos MJ, Hofman A, Koudstaal PJ, Breteler MMB, Stricker BHC. Cyclooxygenase selectivity of nonsteroidal anti-inflammatory drugs and risk of stroke. Arch Intern Med. 2008;168(11):1219-1224.

Hadi NA, Giouvanoudi A, Morton R, Horton PW, Spyrou NM. Variations in gastric emptying times of three stomach regions for simple and complex meals using scintigraphy. IEEE Transactions on Nuclear Science. 2002;49(5):2328-.

Hassid A, Dunn MJ. Microsomal prostaglandin biosynthesis of human kidney. J Biol Chem. 1980;255(6):2472-2475.

Hightower LE. Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides. J Cell Physiol. 1980;102:407-427.

Horton EW, Main IHM, Thompson CJ, Wright PM. Effect of orally administered prostaglandin E1 on gastric secretion and gastrointestinal motility in man. Gut. 1968;9:655-658.

Hoshino T, Tsutsumi S, Tomisato W, Hwang HJ, Tsuchiya T, Mizushima T. Prostaglandin E2 protects gastric mucosal cells from apoptosis via EP2 and EP4 receptor activation. J Biol Chem. 2003;278(15):12752-12758.

Hsieh M, Roth R, Davis DL, Larrabee H, Callaway CW. Hyponatremia in runners requiring on-site medical treatment at a single marathon. Med Sci Sport Exerc. 2002;34(2):185-189.

197

Hudson GM, Green JM, Bishop PA, Richardson MT. Effects of caffeine and aspirin on light resistance training performance, perceived exertion, and pain perception. J Strength Cond Res. 2008;22(6):1950-7.

Huott MA, Storrow AB. A survey of adolescents’ knowledge regarding toxicity of over- the-counter medications. Acad Emerg Med. 1997;4:214-218.

Hurley JC. Endotoxemia: methods of detection and clinical correlates. Clin Microbiol Rev. 1995;8(2):268-292.

Jaattela M, Wissing D. Heat-shock proteins protect cells from monocyte cytotoxicity: possible mechanism of self-protection. J Exp Med. 1993;177:231-236.

Jacobson ED, Bass DE. Effects of sodium salicylate on physiological responses to work in heat. J Appl Phys. 1964;19(1):33-36.

Jacquier-Sarlin MR, Polla BS. Dual regulation of heat-shock transcription factor (HSF) activation and DNA-binding activity by H2O2: role of thioredoxin. Biochem J. 1996;318(187-193).

Jeukendrup AE, Vet-Joop K, Sturk A, et al. Relationship between gastro-intestinal complaints and endotoxaemia, cytokine release and the acute-phase reaction during and after a long-distance triathlon in highly trained men. Clin Sci. 2000;98:47-55.

Johnson AL, Criddle LM. Pass the salt: indications for and implications of using hypertonic saline. Crit Care Nur. 2004;24(5):36-48.

Karim SMM, Carter DC, Bhana D, Adaikan Ganesan P. Effect of orally administered prostaglandin E2 15-methyl analogues on gastric secretion. Br Med J. 1973;1:143-146.

Kauffman GLJ, Reeve JJJ, Grossman MI. Gastric bicarbonate secretion: effect of topical and intravenous 16,16-dimethyl prostaglandin E2. Am J Physiol Gastrointest Liver Physiol. 1980;239(1):G44-G48.

Kawabe J, Ushikubi F, Hasebe N. Prostacyclin in vascular diseases. Circ J. 2010;74(5):836-843.

Keogh JP, Allen A, Garner A. Relationship between gastric mucus synthesis, secretion and surface gel erosion measured in amphibian stomach in vitro E. Clin Exp Pharmacol Physiol. 1997;24:844-849.

Keren G, Epstein Y, Magazanik A. Temporary heat intolerance in a heatstroke patient. Aviat Space Envir Med. 1981;52(2):116-117.

Kilburn KH. Muscular origins of elevated plasma potassium during muscular exercise. J Appl Phys. 1966;21:675-678.

198

Kim H-S, Rhim H, Jeong S-W, Kim JW, Kim I-K. Induction of apoptosis dependent on caspase activities and growth arrest in HL-60 cells by PGA2. Prostaglandins Other Lipid Mediat. 2002;70:169-183.

Kim S, Wook Joo K. Electrolyte and acid-base disturbances associated with non-steroidal anti-inflammatory drugs. Electrolyte Blood Press. 2007;5:116-125.

Knights KM, Mangoni AA, Miners JO. Defining the COX inhibitor selectivity of NSAIDs: implications for understanding toxicity. Expert Rev Clin Pharmacol. 2010;3(6):769-776.

Knochel JP. Enivonrmental heat illness: an eclectic review. Arch Intern Med. 1974;133:841-864.

Kondělková K, Vokurková D, Krejsek J, Borská L, Fiala Z, Andrýs C. Regulatory T cells (Treg) and their roles in immune system with respect to immunopathological disorders Acta Medica. 2010;53(2):73-77.

Konturek SJ, Robert A, Hanchar AJ, Nezamis JE. Comparison of prostacyclin and prostaglandin E2 on gastric secretion, gastrin release, and mucosal blood flow in dogs. Dig Dis Sci. 1980;25(9):673-679.

Kregel KC. Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J Appl Phys. 2002;92(5):2177-2186.

Lafrance JP, Miller DR. Dispensed selective and nonselective nonsteroidal anti- inflammatory drugs and the risk of moderate to severe hyperkalemia: a nested case-control study. Am J Kidney Dis. 2012;60(1):82-89.

Laine L, Connors LG, Reicin A, et al. Serious lower gastrointestinal clinical events with nonselective NSAID or coxib use. Gastroenterology. 2003;124(2):288-292.

Laine L, Curtis SP, Langman M, et al. Lower gastrointestinal events in a double-blind trial of the cyclo-oxygenase-2 selective inhibitor etoricoxib and the traditional nonsteroidal anti-inflammatory drug diclofenac. Gastroenterology. 2008;135(5):1517-1525.

Lam NYL, Rainer TH, Chiu RWK, Lo YMD. EDTA is a better anticoagulant than heparin or citrate for delayed blood processing for plasma DNA analysis. Clin Chem. 2004;50(1):256-257.

Lambert GP, Boylan M, Laventure JP, Bull A, Lanspa S. Effect of aspirin and ibuprofen on GI permeability during exercise. Int J Sports Med. 2007;28(9):722-726.

Lambert GP, Broussard LJ, Mason BL, Mauermann WJ, Gisolfi CV. Gastrointestinal permeability during exercise: effects of aspirin and energy-containing beverages. J Appl Physiol. 2001;90(6):2075-80.

Lambert GP, Lang J, Bull A, et al. Fluid restriction during running increases GI permeability. Int J Sports Med. 2008;29(3):194-198.

199

Lambert GP, Murray R, Eddy D, Scott W, Laird R, Gisolfi CV. Intestinal permeability following the 1998 Ironman Triathlon. Med Sci Sport Exerc. 1999;31(5):S318.

Lambert GP, Schmidt A, Schwarzkopf K, Lanspa S. Effect of aspirin dose on gastrointestinal permeability. Int J Sports Med. 2012;33(6):421-425.

Lambert GP. Role of gastrointestinal permeability in exertional heatstroke. Exerc Sport Sci Rev. 2004;32(4):185-190.

Lambert GP. Stress-induced gastrointestinal barrier dysfunction and its inflammatory effects. J Anim Sci. 2009;87(14 Suppl):E101-E108.

Landry J, Bernier D, Chretien P, Nicole LM, Tanguay RM, Marceau N. Synthesis and degradation of heat shock proteins during development and decay of thermotolerance. Cancer Res. 1982;42:2457-2461.

Laporte J-R, Ibanez L, Vidal X, Vendrell L, Leone R. Upper gastrointestinal bleeding associated with the use of NSAIDs: newer versus older agents. Drug Saf. 2004;27(6):411- 20.

Lewis SC, Langman MJS, Laporte J-R, Matthews JNS, Rawlins MD, Wiholm B-E. Dose– response relationships between individual nonaspirin nonsteroidal anti-inflammatory drugs (NANSAIDs) and serious upper gastrointestinal bleeding: a meta-analysis based on individual patient data. Br J Clin Pharmacol. 2002;54:320-326.

Li H, Edin ML, Gruzdev A, et al. Regulation of T helper cell subsets by cyclooxygenases and their metabolites. Prostaglandins Other Lipid Mediat. 2013;104-105:74-83.

Lichtenberger LM, Zhou Y, Jayaraman V, et al. Insight into NSAID-induced membrane alterations, pathogenesis and therapeutics: characterization of interaction of NSAIDs with phosphatidylcholine. Biochim Biophys Acta. 2012;1821(7):994-1002.

Lim CL, Mackinnon LT. The roles of exercise-induced immune system disturbances in the pathology of heat stroke : the dual pathway model of heat stroke. Sports Med. 2006;36(1):39-64.

Madara JL. Migration of neutrophils through epithelial monolayers. Trends Cell Bio. 1994;4(1):4-7.

Mannion BA, Weiss J, Elsbach P. Separation of sublethal and lethal effects of the bactericidal/permeability increasing protein on Escherichia coli. J Clin Invest. 1990;85(3):853-860.

Marieb EN. Human Anatomy and Physiology. 5th ed. San Francisco, CA: Benjamin Cummings; 2001.

200

Markov AG, Veshnyakova A, Fromm M, Amasheh M, Amasheh S. Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine. J Comp Physiol B. 2010;180(4):591-598.

Marshall JC. The gut as a potential trigger of exercise-induced inflammatory responses. Can J Physiol Pharmacol. 1998;76:479-484.

Martich GD, Danner RL, Ceska M, Suffredini AF. Detection of interleukin 8 and tumor necrosis factor in normal humans after intravenous endotoxin: the effects of antiinflammatory agents. J Exp Med. 1991;173:1021-1024.

Martin JG, Buono MJ. Oral contraceptives elevate core temperature and heart rate during exercise in the heat. Clin Physiol. 1997;17:401-8.

Masso Gonzalez EL, Patrignani P, Tacconelli S, Garcia Rodriguez LA. Variability among nonsteroidal antiinflammatory drugs in risk of upper gastrointestinal bleeding. Arthritis Rheum. 2010;62(6):1592-1601.

Mauger AR, Taylor L, Harding C, Wright B, Foster J, Castle PC. Acute acetaminophen (paracetamol) ingestion improves time to exhaustion during exercise in the heat. Exp Physiol. 2014;99(1):164-71.

Maxton DG, Bjarnason I, Reynolds AP, Catt SD, Peters TJ, Menzies IS. Lactulose 51Cr- labelled ethylenediaminetetra-acetate, L-rhamnose and polyethyleneglycol 500 as probe markers for assessment in vivo of human intestinal permeability. Clin Sci. 1986;71:71-80.

Maxwell NS, Gardner F, Nimmo MA. Intermittent running: muscle metabolism in the heat and effect of hypohydration. Med Sci Sport Exerc. 1999;31(5):675-683.

McAnulty SR, Owens JT, McAnulty LS, et al. Ibuprofen use during extreme exercise: effects on oxidative stress and PGE2. Med Sci Sports Exerc. 2007;39(7):1075-1079.

McArdle WD, Katch FI, Katch VL. Exercise Physiology: Energy, Nutrition, and Human Performance. Fifth ed. Baltimore, MD: Lippincott Williams & Wilkins; 2001.

McCabe III ME, Peura DA, Kadakia SC, Bocek Z, Johnson LF. Gastrointestinal blood loss associated with running a marathon. Digest Dis Sci. 1986;31(11).

McGiff JC, Crowshaw K, Terragno NA, Lonigro AJ. Release of a prostaglandin-like substance into renal venous blood in response to angiotensin II. Cir Res. 1970;26:121-130.

McGiff JC, Terragno NA, Malik KU, Lonigro AJ. Release of a prostaglandin E-like substance from canine kidney by bradykinin. Cir Res. 1972;31:36-43.

McKenney MA, Miller KC, Deal JE, Garden-Robinson JA, Rhee YS. Plasma and electrolyte changes in exercising humans after ingestion of multiple boluses of pickle juice. J Athl Train. 2015;50(2):141-146.

201

McQueen S, Hutton D, Allen A, Garner A. Gastric and duodenal surface mucus gel thickness in rat: effects of prostaglandins and damaging agents. Am J Physiol Gastrointest Liver Physiol. 1983;245:G388-G393.

Mersereau WA, Hinchey EJ. Role of gastric mucosal folds in formation of focal ulcers in the rat. Surgery. 1982;91(2):150-155.

Michie HR, Manogue KR, Spriggs DR, et al. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med. 1988;318:1481-1486.

Mimura Y, Sakisaka S, Harada M, Sata M, Tanikawa K. Role of hepatocytes in direct clearance of lipopolysaccharide in rats. Gastroenterol. 1995;109:1969-1976.

Mokhtare M, Valizadeh SM, Emadian O. Lower gastrointestinal bleeding due to nonsteroid anti-inflammatory drug-induced colopathy case report and literature review. Middle East J Dig Dis. 2013;5:107-111.

Moncada S, Vane JR. Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane A2, and prostacyclin. Pharmacol Rev. 1978;30(3):293-331.

Morita I. Distinct functions of COX-1 and COX-2. Prostaglandins Other Lipid Mediat. 2002;68-69:165-175.

Moses FM. The effect of exercise on the gastrointestinal tract. Sports Med. 1990;9(3):159- 72.

Mosser DD, Martin LH. Induced thermotolerance to apoptosis in human T lymphocyte cell line. J Cell Physiol. 1992;151:561-570.

Muller E, Munker R, Issels R, Wilmanns W. Interaction between tumor necrosis factor-a and HSP70 in human leukemia cells. Leukemia Res. 1993;17(6):523-526.

Musch MW, Ciancio MJ, Sarge K, Chang EB. Induction of heat shock protein 70 protects intestinal epithelial IEC-18 cells from oxidant and thermal injury. Am J Physiol Cell Physiol. 1996;270:C429-C436.

Nakahura T, Griswold W, Lemire J, Mendoza S, Rezni V. Nonsteroidal anti-inflammatory drug use in adolescence. J Adolescent Health. 1998;23:307-310.

Naproxen Sodium. National Center for Biotechnology Information. https://pubchem.ncbi.nlm.nih.gov/compound/23681059. Accessed March 13, 2016.

Nelson DL, Cox MM. Lehninger Principles of Biochemistry. 5th ed. New York, NY: W. H. Freeman and Company; 2008.

Nickells RW, Browder LW. A role for glyceraldehyde-3-phosphate dehydrogenase in the development of thermotolerance in Xenopus laevis embryos. Dev Biol. 1985;112:391-395.

202

Nieman DC, Henson DA, Dumke CL, et al. Ibuprofen use, endotoxemia, inflammation, and plasma cytokines during ultramarathon competition. Brain Behav Immun. 2006;20(6):578-584.

Noakes TD, Sharwood K, Speedy D, et al. Three independent biological mechanisms cause exercise-associated hyponatremia: evidence from 2,135 weighed competitive athletic performances. Proc Natl Acad Sci U S A. 2005;102(51):18550-18555.

Noda M, Kariura Y, Pannasch U, et al. Neuroprotective role of bradykinin because of the attenuation of pro-inflammatory cytokine release from activated microglia. J Neurochem. 2007;101(2):397-410.

Northridge DB, Grant S, Ford I, et al. Novel exercise protocol suitable for use on a treadmill or a bicycle ergometer. Br Heart J. 1990;64:313-316.

Nusrat A, Turner JR, Madara JL. Molecular physiology and pathophysiology of tight junctions IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Am J Physiol Gastrointest Liver Physiol. 2000;279:G851-G857.

O'Neill S, Ingman TG, Wigmore SJ, Harrison EM, Bellamy CO. Differential expression of heat shock proteins in healthy and diseased human renal allografts. Ann Transplant. 2013;18:550-557.

Oort FA, Terhaar Sive Droste JS, Van Der Hulst RW, et al. Colonoscopy-controlled intra- individual comparisons to screen relevant neoplasia: faecal immunochemical test vs. guaiac-based faecal occult blood test. Aliment Pharmacol Ther. 2010;31(3):432-439.

Ostrowski K, Rohde T, Zacho M, Asp S, Pedersen BK. Evidence that interleukin-6 is produced in human skeletal muscle during prolonged running. J Physiol. 1998;508(3):949- 953.

OTC sales by category 2011-2014. Consumer Healthcare Products Association2015.

Pairet M, van Ryn J. Experimental models used to investigate the differential inhibition of cyclooxygenase-1 and cyclooxygenase-2 by non-steroidal anti-inflammatory drugs. Inflamm Res. 1998;47(S2):S93-S101.

Pals KL, Chang RT, Ryan AJ, Gisolfi CV. Effect of running intensity on intestinal permeability. J Appl Physiol. 1997;82(2):571-576.

Parks DA, Bulkley GB, Granger DN. Role of oxygen-derived free radicals in digestive tract diseases. Surgery. 1983;94(3):415-422.

PAR-Q & YOU. In: Physiology CSfE, ed2002.

Patrignani P, Filabozzi P, Patrono C. Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects. J Clin Invest. 1982;69:1366-1372.

203

Patrono C, Garcia Rodriguez LA, Landolfi R, Baigent C. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med. 2005;353:2373-2383.

Pedersen BK, Rohde T, Ostrowski K. Recovery of the immune system after exercise. Acta Physiol Scand. 1998;162:325-32.

Pedersen BK, Toft AD. Effects of exercise on lymphocytes and cytokines. Br J Sports Med. 2000;34:246-251.

Pernerstorfer T, Schmid R, Bieglmayer C, Eichler H-G, Kapiotis S, Jilma B. Acetaminophen has greater antipyretic efficacy than aspirin in endotoxemia: a randomized, double-blind, placebo-controlled trial. Clin Pharmacol Ther. 1999;66:51-57.

Pescatello LS, ed ACSM's Guidelines for Exercise Testing and Prescription. 9th ed. Baltimore, MD: Lippincott Williams & Wilkins; 2014. Arena R, ed. Exercise Testing.

Peters HPF, Bos M, Seebregts L, et al. Gastrointestinal symptoms in long-distance runners, cyclists, and triathletes: prevalence, medication, and etiology. Am J Gastroenterol. 1999;94(6):1570-81.

Peters HPF, Wiersma WC, Akkermans LM, et al. Gastrointestinal mucosal integrity after prolonged exercise with fluid supplementation. Med Sci Sport Exerc. 2000;32(1):134-142.

Peters HPF, Wiersma WC, Koerselman J, et al. The effect of a sports drink on gastroesophageal reflux during a run-bike-run test. Int J Sports Med. 1999;20:65-70.

Pfeiffer B, Cotterill A, Grathwohl D, Stellingwerff T, Jeukendrup AE. The effect of carbohydrate gels on gastrointestinal tolerance during a 16-km run. Int J Sport Nutr Exerc Metab. 2009;19:485-503.

Pfeiffer B, Stellingwerff T, Hodgson AB, et al. Nutritional intake and gastrointestinal problems during competitive endurance events. Med Sci Sports Exerc. 2012;44(2):344- 351.

Pivarnik JM, Marichal CJ, Spillman T, Morrow Jr. JR. Menstrual cycle phase affects temperature regulation during endurance exercise. J Appl Phys. 1992;72(2):543-8.

Polla BS, Bachelet M, Elia G, Santoro MG. Stress proteins in inflammation. Ann N Y Acad Sci. 1998;851(1):75-85.

Popowski RA, Oppliger RA, Lambert GP, Johnson RF, Johnson AK, Gisolfi CV. Blood and urinary measures of hydration status during progressive acute dehydration. Med Sci Sport Exerc. 2001;33(5):747-753.

Preparticipation Physical Examination Form. In: Medicine AMSfS, ed2010.

Prostanoids - prostaglandins, prostacyclins, and thromboxanes. AOCS Lipid Library; 2014. Accessed 5/12/2015.

204

Proulx CI, Ducharme MB, Kenny GP. Safe cooling limits from exercise-induced hyperthermia. Eur J Appl Physiol. 2006;96(4):434-445.

Ramwell PW, Leovey EMK, Sintetos AL. Regulation of the arachidonic acid cascade. Bio Reprod. 1977;16:70-87.

Rao R. Endotoxemia and gut barrier dysfunction in alcoholic liver disease. Hepatology. 2009;50(2):638-644.

Rehrer N, Smets A, Reynaert H, Goes E, de Meirleir K. Effect of exercise on portal vein blood flow in man. Med Sci Sport Exerc. 2001;33(9):1533-1537.

Rehrer NJ, Beckers EJ, Brouns F, Ten Hoor F, Saris WHM. Effects of dehydration on gastric emptying and gastrointestinal distress while running. Med Sci Sports Exerc. 1990;22(6):790-795.

Rehrer NJ, Meijer GA. Biomechanical vibration of the abdominal region during running and bicycling. J Sports Med Phys Fitness. 1991;31(2):231-234.

Rhind SG, Gannon GA, Shephard RJ, Shek PN. Indomethacin modulates circulating cytokine responses to strenuous exercise in humans. Cytokine. 2002;19(3):153-158.

Rhind SG, Gannon GA, Suzui M, Shephard RJ, Shek PN. Indomethacin inhibits circulating PGE2 and reverses postexercise suppression of natural killer cell activity. Am J Physiol Regul Integr Comp Physiol. 1999;276:R1496-R1505.

Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol. 2011;31(5):986-1000.

Roberts WO. Exertional heat stroke during a cool weather marathon: a case study. Med Sci Sports Exerc. 2006;38(7):1197-1203.

Robertson RJ, Moyna NM, Sward KL, Millich NB, Goss FL, Thompson PD. Gender comparison of RPE at absolute and relative physiological criteria. Med Sci Sport Exerc. 2000;32(12):2120-9.

Robinson TA, Hawley JA, Palmer GS, et al. Water ingestion does not improve 1-h cycling performance in moderate ambient temperatures. Eur J Appl Physiol. 1995;71:153-160.

Roi GS, Garagiola U, Verza P, et al. Aspirin does not affect exercise performance. Int J Sports Med. 1994;15(5):224-227.

Rosner MH, Kirven J. Exercise-associated hyponatremia. Clin J Am Soc Nephrol. 2007;2(1):151-161.

Rubin GM, Hogness DS. Effect of heat shock on the synthesis of low molecular weight RNAs in Drosophila: accumulation of a novel form of 5S RNA. Cell. 1975;6:207-213.

205

Ryan AJ, Chang R-T, Gisolfi CV. Gastrointestinal permeability following aspirin intake and prolonged running. Med Sci Sport Exerc. 1996;28(6):698-705.

Samali A, Cotter TG. Heat shock proteins increase resistance to apoptosis. Exp Cell Res. 1996;223:163-170.

Santoro MG, Garaci E, Amici C. Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells. Proc Natl Acad Sci U S A. 1989;86(21):8407-8411.

Santoro MG. Antiviral activity of cyclopentenone prostanoids. Trends Microbiol. 1997;5(7):276-281.

Santoro MG. Heat shock factors and the control of the stress response. Biochem Pharmcol. 2000;59(1):55-63.

Saverymuttu SH, Thomas A, Grundy A, Maxwell JD. Ileal stricturing after long term indomethacin treatment. Postgrad Med J. 1986;62:267-268.

Sawka MN, Burke LM, Eichner ER, Maughan RJ, Montain SJ, Stachenfeld NS. American College of Sports Medicine position stand. Exercise and fluid replacement. Med Sci Sport Exerc. 2007;39(2):377-390.

Sawka MN, Wenger CB. Physiological responses to acute exercise-heat stress. Natick, MA: U.S. Army Research Institute of Environmental Medicine; 1988.

Schafer AI. Effects of nonsteroidal antiinflammatory drugs on platelet function and systemic hemostasis. J Clin Pharmacol. 1995;35:209-219.

Scotney B, Reid S. Body weight, serum sodium levels, and renal function in an ultra- distance mountain run. Clin J Sport Med. 2015;25:341-346.

Sears CL. A dynamic partnership: celebrating our gut flora. Anaerobe. 2005;11(5):247- 251.

Sepehri G, Pourranjbar M, Moshtaghi-Kashanian G-R, Khachaki AS, Sepehri E. The effect of a prophylactic dose of ibuprofen on plasma level of interleukin 1, interleukin 6 and tumor necrosis factor-alpha in a 1500 m running practice. Am J Appl Sci. 2011;8(1):50-54.

Shah AA, Thjodleifsson B, Murray FE, et al. Selective inhibition of COX-2 in humans is associated with less gastrointestinal injury: a comparison of nimesulide and naproxen. Gut. 2001;48:339-346.

Shibata M, Ohkubo T, Takahashi H, Inoki R. Interaction of bradykinin with substance P on vascular permeability and pain response. Japan J Pharmacol. 1986;41:427-429.

Shibolet S, Coll R, Gilat T, Sohar E. Heatstroke: its clinical picture and mechanism in 36 cases. Q J Med. 1967;36(144):525-548.

206

Smecuol E, Bai JC, Sugai E, et al. Acute gastrointestinal permeability responses to different non-steroidal anti-inflammatory drugs. Gut. 2001;49:6.

Snyder YM, Guthrie L, Evans GF, Zuckerman SH. Transcriptional inhibition of endotoxin- induced monokine synthesis following heat shock in murine peritoneal macrophages. J Leukoc Biol. 1992;51:181-187.

Sodium Salicylate. National Center for Biotechnology Information. https://pubchem.ncbi.nlm.nih.gov/compound/16760658 Accessed March 13, 2016.

Soll AH. Specific inhibition by prostaglandins E2 and 12 of histamine-stimulated [14C]aminopyrine accumulation and cyclic adenosine monophosphate generation by isolated canine parietal cells. J Clin Invest. 1980;65:1222-1229.

Spinas GA, Bloesch D, Keller U, Zimmerli W, Cammisuli S. Pretreatment with ibuprofen augments circulating tumor necrosis factor-a, interleukin-6, and elastase during acute endotoxinemia. J Infect Dis. 1991;163(1):89-95.

Starkie RL, Hargreaves M, Rolland J, Febbraio MA. Heat stress, cytokines, and the immune response to exercise. Brain Behav Immun. 2005;19(5):404-412.

Steensberg A, van Hall G, Osada T, Sacchetti M, Saltin B, Pedersen BK. Production of interleukin-6 in contracting human skeletal muscles can account for the exercise-induced increase in plasma interleukin-6. J Physiol. 2000;529(1):237-42.

Stevens A. Update: Douglas County football player has died. The Atlanta Journal- Constitution. August 11, 2014.

Stokes JB. Integrated actions of renal medullary prostaglandins in the control of water excretion. Am J Physiol Renal Fluid Electrolyte Physiol. 1981;240:F471-F480.

Straus DS, Glass CK. Cyclopentenone prostaglandins: new insights on biological activities and cellular targets. Med Res Rev. 2001;21(3):185-210.

Straus DS, Pascual G, Li M, et al. 15-deoxy-delta 12,14-prostaglandin J2 inhibits multiple steps in the NF-kappa B signaling pathway. Proc Natl Acad Sci U S A. 2000;97(9):4844- 4849.

Stringer v. Minnesota Vikings Football Club LLC. Supreme Court of Minnesota;2005.

Stuempfle KJ, Hoffman MD, Hew-Butler T. Association of gastrointestinal distress in ultramarathoners with race diet. Int J Sport Nutr Exerc Metab. 2013;23:103-109.

Suenaert P, Bulteel V, Lemmens L, et al. Anti-tumour necrosis factor treatment restores the gut barrier in Chohn's disease. Am J Gastroenterol. 2002;97(8):2000-2004.

Suzic Lazic J, Dikic N, Radivojevic N, et al. Dietary supplements and medications in elite sport--polypharmacy or real need? Scand J Med Sci Sports. 2011;21(2):260-267.

207

Suzuki K, Araki H, Mizoguchi H, Furukawa O, Takeuchi K. Prostaglandin E inhibits indomethacin-induced gastric lesions through EP-1 receptors. Digestion. 2001;63:92-101.

Suzuki K, Peake J, Nosaka K, et al. Changes in markers of muscle damage, inflammation and HSP70 after an Ironman Triathlon race. Eur J Appl Physiol. 2006;98(6):525-534.

Tacconelli S, Capone ML, Patrignani P. Clinical pharmacology of novel selective COX-2 inhibitors. Curr Pharm Des. 2004;10:589-601.

Takahashi S, Takeuchi K, Okabe S. EP4 receptor mediation of prostaglandin E2-stimulated mucus secretion by rabbit gastric epithelial cells. Biochem Pharmcol. 1999;58:1997-2002.

Takeuchi K, Kita K, Hayashi S, Aihara E. Regulatory mechanism of duodenal bicarbonate secretion: Roles of endogenous prostaglandins and nitric oxide. Pharmacol Ther. 2011;130(1):59-70.

Takeuchi K, Ueshima K, Hironaka Y, Fujiokaa Y, Matsumoto J, Okabe S. Oxygen free radicals and lipid peroxidation in the pathogenesis of gastric mucosal lesions induced by indomethacin in rats. Digestion. 1991;49(3):175-784.

Takeuchi K. Pathogenesis of NSAID-induced gastric damage: importance of cyclooxygenase inhibition and gastric hypermotility. World J Gastroenterol. 2012;18(18):2147-2160.

Tazuke Y, Drongowski RA, Teitelbaum DH, Coran AG. Interleukin-6 changes tight junction permeability and intracellular phospholipid content in a human enterocyte cell culture model. Pediatr Surg Int. 2003;19(5):321-325. ter Steege RW, Kolkman JJ. Review article: the pathophysiology and management of gastrointestinal symptoms during physical exercise, and the role of splanchnic blood flow. Aliment Pharmacol Ther. 2012;35(5):516-528.

Timmons BW, Hamadeh MJ, Devries MC, Tarnopolsky MA. Influence of gender, menstrual phase, and oral contraceptive use on immunological changes in response to prolonged cycling. J Appl Physiol (1985). 2005;99(3):979-85.

Todd PA, Clissold SP. Naproxen. A reappraisal of its pharmacology, and therapeutic use in rheumatic diseases and pain states. Drugs. 1990;40(1):91-137.

Todryk S, Melcher AA, Hardwick N, et al. Heat shock protein 70 induced during tumor cell killing induces Th1 cytokines and targets immature dendritic cell precursors to enhance antigen uptake. J Immunol. 1999;163:1398-1408.

Top 10 OTC brands by revenue in the U.S. in 2013. The Statistical Portal2013.

Toyokuni S. Reactive oxygen species-induced molecular damage and its application in pathology. Pathol Int. 1999;49:91-102.

208

Tracey KJ, Fong Y, Hesse DG, et al. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature. 1987;330:662-664.

Treating pain with NSAID medications Consumer Reports2013.

Tscholl P, Alonso JM, Dolle G, Junge A, Dvorak J. The use of drugs and nutritional supplements in top-level track and field athletes. Am J Sports Med. 2010;38(1):133-140.

Tsutsumi S, Haruna R, Tomisato W, et al. Effects of prostaglandins on spontaneous apoptosis in gastric mucosal cells. Dig Dis Sci. 2002;47(1):84-89.

Turner JR, Rill BK, Carlson SL, et al. Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation. Am J Physiol Cell Physiol. 1997;273:C1378-C1385.

Turner JR. Intestinal mucosal barrier function in health and disease. Nat Rev Immunol. 2009;9(11):799-809.

Turner JR. 'Putting the squeeze' on the tight junction: understanding cytoskeletal regulation. Semin Cell Dev Biol. 2000;11(4):301-308.

Ueki S, Takeuchi K, Okabe S. Gastric motility is an important factor in the pathogenesis of indomethacin-induced gastric mucosal lesions in rats. Dig Dis Sci. 1988;33(2):209-216.

Ukabam SO, Cooper BT. Small intestinal permeability to mannitol, lactulose, and polyethylene glycol 400 in celiac disease. Dig Dis Sci. 1984;29:809-816.

van Nieuwenhoven MA, Brouns F, Brummer RJ. Gastrointestinal profile of symptomatic athletes at rest and during physical exercise. Eur J Appl Physiol. 2004;91(4):429-434.

van Nieuwenhoven MA, Brouns F, Brummer RJ. The effect of physical exercise on parameters of gastorintestinal function. Neurogastroenterol Mot. 1999;11:431-439.

van Wijck K, Verlinden TJ, van Eijk HM, et al. Novel multi-sugar assay for site-specific gastrointestinal permeability analysis: a randomized controlled crossover trial. Clin Nutr. 2013;32(2):245-251.

Vane JR, Botting RM. Anti-inflammatory drugs and their mechanism of action. Inflamm Res. 1998;47:S78-S87.

Vane JR. Differential inhibition of cyclooxygenase isoforms an explanation of the action of NSAIDs. J Clin Rheumatol. 1998;4:S3-S10.

Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature. 1971;231(25):232-235.

Vivier E, Raulet DH, Moretta A, et al. Innate or adaptive immunity? The example of natural killer cells. Science. 2011;331(6013):44-49.

209

Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol. 2008;9(5):503-510.

Voet D, Voet JG. Biochemistry. Hoboken, NJ: John Wiley & Sons, Inc.; 2011.

Walch M, Dotiwala F, Mulik S, et al. Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes. Cell. 2014;157(6):1309-1323.

Wallace JL, Granger DN. Pathogenesis of NSAID gastropathy: are neutrophils the culprits? Trends Pharmacol Sci. 1992;13(4):129-131.

Wallace JL. Prostaglandins, NSAIDs, and gastric mucosal protection: why doesn't the stomach digest itself? Physiol Rev. 2008;88:1547-1565.

Warden SJ. Prophylactic use of NSAIDs by athletes: a risk/benefit assessment. Phys Sportsmed. 2010;1(38):1-7.

Warner DC, Schnepf G, Barrett MS, Dian D, Swigonski NL. Prevalence, attitudes, and behaviors related to the use of nonsteroidal anti-inflammatory drugs (NSAIDs) in student athletes. J Adolescent Health. 2002;30:150-153.

Warner TD, Giuliano F, Vojnovic I, Bukasa A, Mitchell JA, Vane JR. Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis. Proc Natl Acad Sci U S A. 1999;96:7563-7568.

Waterman JJ, Kapur R. Upper gastrointestinal issues in athletes. Curr Sports Med Rep. 2012;11(2):99-104.

Way L, Durbin RP. Inhibition of gastric acid secretion in vitro by prostaglandin E1. Nature. 1969;221:874-875.

Welc SS, Clanton TL, Dineen SM, Leon LR. Heat stroke activates a stress-induced cytokine response in skeletal muscle. J Appl Phys. 2013;115:1126-1137.

Welch WJ, Suhan JP. Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment. J Cell Biol. 1985;101(4):1198-1211.

Welch WJ. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol Rev. 1992;72(4):1063-1081.

Wemple RD, Morocco TS, Mack GW. Influence of sodium replacement on fluid ingestion following exercise-induced dehydration. Int J Sport Nutr. 1997;7:104-116.

Wenner MM, Stachenfeld NS. Blood pressure and water regulation: understanding sex hormone effects within and between men and women. J Physiol. 2012;590(23):5949-5961.

210

Wharam PC, Speedy DB, Noakes TD, Thompson JM, Reid SA, Holtzhausen LM. NSAID use increases the risk of developing hyponatremia during an Ironman triathlon. Med Sci Sport Exerc. 2006;38(4):618-622.

Young GP, Cole S. New stool screening tests for colorectal cancer. Digestion. 2007;76(1):26-33.

Young GP, St. John DJB, Winawer SJ, Rozen P. Choice of fecal occult blood tests for colorectal cancer screening: recommendations based on performance characteristics in population studies. Am J Gastroenterol. 2002;97(10):2499-2507.

Zaslavsky BY, Baevskii AV, Rogozhin SV, et al. Relative hydrophobicity of synthetic macromolecules. I. Polyethylene glycol, polyacrylamide, and polypyrrolidone J Chromatogr. 1985;285:63-68.

Zenewicz LA, Flavell RA. Recent advances in IL-22 biology. Int Immunol. 2011;23(3):159-163.

Zushi S, Shinomura Y, Kiyohara T, et al. Role of prostaglandins in intestinal epithelial restitution stimulated by growth factors. Am J Physiol Gastrointest Liver Physiol. 1996;270:G757-G762.

211 APPENDIX A. INFORMED CONSENT FORM

Title of the Study: Non-steroidal Anti-inflammatory Drugs on Gastrointestinal Permeability during Thermal Stress in Exercising Humans

Investigators: Dawn M. Emerson, MS, ATC; J. Mark Davis, PhD; Toni M. Torres- McGehee, PhD, ATC; Stephen Chen, PhD

The purpose of this study is to determine the effects of the non-steroidal anti-inflammatory drug (NSAID) Naproxen on core body temperature (Tc), gastrointestinal (GI) permeability, inflammatory markers, and fluid regulation during exercise in a thermal environment. The study is being conducted to fulfill partial requirements for a Doctorate degree in exercise science - applied physiology. You are being invited to participate in this study because you are between the ages of 18 and 38yrs, endurance trained, and are in good general health. The study will take place at the Exercise Science Laboratories at the University of South Carolina’s Arnold School of Public Health. If you agree to participate, you will be participating with approximately 20 other people.

STUDY PARTICIPATION After signing the informed consent form, potential participants will complete a Health and Injury History Questionnaire. Any potential participant with a health or safety concern will be required to have his/her questionnaire reviewed by the physician overseeing this study to determine inclusion or exclusion into the study.

Upon acceptance into the study, participants will complete a cycle test to measure VO2max. Participants will then be randomly assigned to complete four trials: control (C), placebo and heat stress (PHeat), Naproxen (Npx), and Naproxen and heat stress (NpxHeat). You will complete all four conditions in a random order with a minimum of 7 days in between each trial. You will be unaware of whether you are taking a placebo or Naproxen. During the course of this study, you will be asked to maintain normal levels of physical activity, normal state of hydration, and avoid changes in caffeine consumption and dehydration-inducing substances such as alcohol. The study will require you to perform a 90 minute cycle time trial and allow Tc, GI permeability, inflammatory markers, and hydration measurements before, during, and after the cycle bout. You will be provided fluids to consume throughout the sessions in order to maintain your hydration. The time commitment for each trial will be approximately 6 hours each time. Total time to complete all trials will be 24 hours.

212

CONDITIONS Control You will be given 3 placebo (no medicine) capsules with Take Home Instructions instructing you when to take each capsule. Upon arriving to the lab you will provide hydration measures, ensure the temperature sensor is working, and have a flexible catheter inserted into a vein in your arm for blood measures. You will complete a 90-minute cycle protocol in a cool environment (15°C/59°F, 30% humidity).

PHeat You will be given 3 placebo (no medicine) capsules with Take Home Instructions instructing you when to take each capsule. Upon arriving to the lab you will provide hydration measures, ensure the temperature sensor is working, and have a flexible catheter inserted into a vein in your arm for blood measures. You will complete a 90-minute cycle protocol in a hot, humid environment (35°C/95°F, 30% humidity).

Npx You will be given 3 over-the-counter doses of Naproxen with Take Home Instructions instructing you when to take each capsule. Upon arriving to the lab you will provide hydration measures, ensure the temperature sensor is working, and have a flexible catheter inserted into a vein in your arm for blood measures. You will complete a 90-minute cycle protocol in a cool environment (15°C/59°F, 30% humidity).

NpxHeat You will be given 3 over-the-counter doses of Naproxen with Take Home Instructions instructing you when to take each capsule. Upon arriving to the lab you will provide hydration measures, ensure the temperature sensor is working, and have a flexible catheter inserted into a vein in your arm for blood measures. You will complete a 90-minute cycle protocol in a hot, humid environment (35°C/95°F, 30% humidity).

INSTRUMENTS AND PROTOCOLS STEEP Test Used to measure VO2max, this cycle test progressively increases in difficulty and respiratory variables are measured throughout. Participants with a VO2max between 50- 60 ml/kg/min for males and 48-58 ml/kg/min for females will be considered endurance trained and qualify to participate in the study.

Naproxen Naproxen (commonly known as Aleve), is an over-the-counter available NSAID. You will receive a 24-hour dosage (3 pills, 220mg per pill) with instructions on what time to take the pills (1 pill every 8 hours).

Placebo During the placebo trials, you will be provided 3 pills that contain no medicine and are made to resemble the Naproxen. You will take 1 pill, every 8 hours.

213

Temperature Measures Temperature will be continuously monitored using a flexible rectal thermometer. Thermometers are attached to a cable which inserts into a machine that records temperature. After inserting the thermometer you will not need to remove it until completion of the session. Minimal discomfort should be felt during exercise.

Blood and Urine Measures To obtain the blood we will draw blood from a vein in your arm prior to the cycling exercise. A flexible catheter will be used so only one needle stick is required and you will complete the exercises. For each blood draw, we will take 1 tube (1.2 tsp). Approximately 9 blood draws will be done during one data collection session for a total of 5 tbsp (1/6th of what you would donate to the American Red Cross).

To obtain urine samples, participants will be provided a cup, and will be able to use a private restroom to collect the urine sample. You will be instructed to completely void your bladder into the cup during each urine collection. The cup will be labeled with your participant number.

GI Permeability, GI Bleeding, and Inflammatory Markers Gastrointestinal permeability will be determined by the presence of ingestible sugar probes in urine samples. Upon arriving to the laboratory you will be given 150ml (about ¾C) of a sugar-water solution to consume. You will then provide urine samples throughout the session for a total of a 5 hour period.

To determine GI bleeding we will measure blood using take-home kits. Prior to each session you will be provide a kit, including instructions, and asked to collect the first stool sample after taking the first pill (Naproxen or placebo). You will then be asked to take the first stool sample following the exercise session. You will then bring the kit to the laboratory. Only your participant number will be used on take-home kits.

Inflammation will be determined by the presence of the inflammatory markers TNF-α and IL-6 in your blood sample.

Hydration and Electrolyte Measures We will determine hydration status and electrolyte balance through urine and blood measures. Hydration measures will include Posm, urine osmolality (Uosm), urine specific gravity (USG) and percent change in body mass (%BM). Electrolytes will be measured from P[Na+] and potassium (P[K+]).

Borg Scale for Rate of Perceived Exertion We will measure your rate of perceived exertion before, every 15 minutes during, and after exercise.

214

Heart Rate and Blood Pressure To ensure participants remain at safe limits and determine any effects from NSAID use, we will continuously measure heart rate. Blood pressure will be measured before, every 15 minutes during, and after exercise.

Gastrointestinal Symptom Index GI distress before and after exercise will be assessed using a symptom questionnaire. The index is divided into 3 sections: 1) upper abdominal problems, 2) lower abdominal problems, and 3) systemic problems.

Cycle Time Trial A 90-minute cycle time trial will be used to determine the participant's performance. Participants will cycle at a steady 70% VO2max for 80 minutes. Any participant that cannot complete this will be disqualified from the study. The last 10 minutes of the cycle trial will be a maximum effort for distance.

Diet and Activity Logs You will be asked to complete an online form to track your diet and activity for 3 days before and 1 day after each data collection session. You will be given a login and password for your personal account.

SECURITY OF PERSONAL INFORMATION Participation will be confidential. To assure your confidentiality, all information pertaining to this study and participation will be stored in a secure and locked cabinet. Additionally, a code number will be assigned to each participant at the beginning of the project. This number will be used on project records rather than your name, and no one other than the researchers will be able to link your information with your name. Data from all participants will be analyzed together. All blood and urine samples will be discarded after analysis. All data will be stored on a secure, password protected computer and only researchers will have access to the data. The results of this study may be reported in professional journals or presented at meetings; however, you will not be identified.

RISKS There is a moderate risk for developing adverse effects due to NSAID use. Potential risks and side effects include: ulcers, bleeding, or holes, which may develop without warning, in the stomach or intestine; constipation; diarrhea; gas; sores in mouth; excessive thirst; headache; dizziness; lightheadedness; drowsiness; difficulty falling asleep or staying asleep; burning or tingling in the arms or legs; cold symptoms; ringing in the ears; hearing problems. Serious side effects include: changes in vision; feeling that the tablet is stuck in your throat; unexplained weight gain; sore throat, fever, chills, and other signs of infection; blisters; rash; skin reddening; itching; hives; swelling of the eyes, face, lips, tongue, throat, arms, hands, feet, ankles, or lower legs; difficulty breathing or swallowing; hoarseness; excessive tiredness; pain in the upper right part of the stomach; nausea; loss of appetite; yellowing of the skin or eyes; flu-like symptoms; bruises or purple blotches under the skin; pale skin; fast heartbeat; cloudy, discolored, or bloody urine; back pain; difficult or painful

215

urination. In the event you experience these symptoms, you may request to stop or the researchers will stop your trial.

There is a low probability that you experience a more serious risk during this study. Serious risks include, but are not limited to, heat illness and cardiac events. There is a risk of developing an exertional heat illness (for example, heat exhaustion and heat stroke) while exercising in a thermal environment. In the event that you experience symptoms of heat exhaustion, (nausea, vomiting, dizziness, etc.) you may request to stop or the researchers will stop your trial. In the case that you are suffering from a heat stroke, your trial will be immediately stopped and ice/cold water immersion will be available. Ice/cold water immersion is the best method available to quickly decrease the body’s Tc to a safe limit. The probability of developing a heat stroke during this study is minimal. We will continuously monitor Tc and cease the cycling protocol if and when you reach a Tc of 40°C (104°F) and/or when you begin to experience any of the symptoms mentioned above.

As with any exercise, there is a small risk of a cardiac event due to an undetected cardiac condition. Heart rate will be monitored continuously and blood pressure will be monitored every 15 minutes to help ensure your safety. All investigators are American Red Cross AED, CPR Professional Rescuer and First Aid certified.

Other risks of this study are general risks associated with exercise, such as muscle or tendon strains. Cycling is a low impact exercise and should minimize the risk of developing a muscle or tendon injury. There is no known risk for consuming the sugar solution.

Risks of drawing blood include temporary discomfort from the needle stick, bruising, tenderness and infection. Fainting could also occur. OSHA guidelines will be followed to minimizing these risks. Investigators will abide by all OSHA guidelines and will be able to act if there is an emergency or other medical issue during the study.

PARTICIPANT SAFETY In the event you experience a heat stroke there will be a cold/ice water tub available for immersion. Ice water immersion is the fastest and safest method to cool the body to a safe Tc. In the event that you experience a sudden stop in your heart, there is access to an Automated External Defribillator (AED), room 316 of the Public Health Research Center. All investigators are trained to use an AED and in CPR. If you experience any medical issues such as a heat illness or cardiac event as a participant in this study, the investigators will provide appropriate immediate care and you will be referred for additional medical treatment if necessary. However, in the event you suffer from a research related injury, there will be no financial compensation from the investigators or the University of South Carolina to assist with medical fees.

The investigators will terminate your participation in the study, without your consent, if you suffer any symptoms or conditions that the investigators believe make it inadvisable for you to continue in the study. In the case of a trial being terminated due to adverse symptoms an incident report will be completed and given to the physician overseeing the study. In the case of trial termination due to mild symptoms (dizziness, nausea, headache,

216

etc.) the participant may choose to continue the trial at another date. However, the participant cannot continue until the physician has reviewed the incident report and deems it appropriate for the participant to continue with the study. In the case of a more severe incident (for example, heat stroke) the participant will not be allowed to continue in the study.

PARTICIPANT PAYMENT As a participant in this study, you will have the opportunity to earn $100.00. Participants will be paid $20.00 at the completion of each trial, for a total of $80.00 ($20.00 x four trials). Upon completion of the fourth trial, each participant will be given $20.00 if he or she follows all study instructions and provides maximum effort during all exercise trials.

BENEFITS Participating in this study will increase your awareness of personal hydration behaviors. Each participant will receive a personalized hydration protocol that includes fluid and electrolyte needs during exercise. The primary investigator will also be available to discuss the individual reports and answer any specific questions the participant has in regards to fluid and electrolyte needs during exercise.

The information from this study will be helpful for competitive athletes as well as recreational athletes and athletic trainers regarding the use of NSAIDs in hot, humid environments.

VOLUNTARINESS Participation is voluntary. You may decide not to participate at all, or to stop participating at any time, for any reason without negative consequences. Participants wishing to withdraw voluntarily from the study should notify the primary investigator (Dawn Emerson) by phone or email. Your participation, non-participation and/or withdrawal will not affect your grades or your relationship with your professors, college(s), or the University of South Carolina.

QUESTIONS If you have any questions, please feel free to ask at any time. You should contact Dawn M. Emerson at (cell) (XXX) XXX-XXXX or (email) [email protected] or Dr. Mark Davis at (office) (XXX) XXX-XXXX or (email) [email protected] if you have any questions or if you believe that you have suffered a research related injury.

If you have any questions about your rights as a research subject contact, Lisa Marie Johnson, IRB Manager, Office of Research Compliance, University of South Carolina, 901 Sumter Street, Byrnes 515, Columbia, SC 29208, Phone: (803) 777-7095 or [email protected]. The Office of Research Compliance is an administrative office that supports the USC Institutional Review Board. The Institutional Review Board (IRB) consists of representatives from a variety of scientific disciplines, non-scientists, and community members for the primary purpose of protecting the rights and welfare of human subjects enrolled in research studies.

217

SIGNATURES I have read the contents of this Consent Form. The purpose of this study, procedures to be followed, risks and benefits have been explained to me. I have been allowed to ask questions, and my questions have been answered to my satisfaction. I have been told whom to contact if I have additional questions. I agree to participate in this study, knowing that I may withdraw at any time. I have been given a copy of this Consent Form to keep for my reference.

______Date ______Signature of Participant Name (Please Print) ------I have explained and defined in detail the research procedure in which the participant has agreed to participate and have offered him a copy of this informed consent form. ______Date ______Signature of Investigator Name (Please Print)

218

APPENDIX B. HEALTH AND INJURY HISTORY QUESTIONNAIRE

Instructions: Complete the following questions the best of your knowledge/ability. Let the investigator know if you need further explanation.

Part 1. Participant Information Name: ______Age: ______Participant #: _____ Part 2. Physical Activity Readiness Questionnaire© (Canadian Society for Exercise Physiology) Yes No 1. Has your doctor ever said that you have a heart condition and that you should only do physical activity recommended by a doctor? 2. Do you feel pain in your chest when you do physical activity?

3. In the past month, have you had chest pain when you were not doing physical activity? 4. Do you lose your balance because of dizziness or do you ever lose consciousness? 5. Do you have a bone or joint problem (for example, back, knee or hip) that could be made worse by a change in your physical activity? 6. Is your doctor currently prescribing drugs (for example, water pills) for your blood pressure or heart condition? 7. Do you know of any other reason why you should not do physical activity?

Part 3. Medical History Explain “yes” answers below. (American Medical Society for Sports Medicine, American Orthopedic Society for Sports Medicine, American Heart Association, and American College of Sports Medicine) Yes No 8. Have you had a medical illness or injury since your last check up or sports physical? 9. Do you have an ongoing chronic illness?

10. Have you ever passed out during or after exercise?

219

11. Have you ever been dizzy or fainted during or after exercise?

12. Have you had a medical illness or injury since your last check up or sports physical? 13. Have you ever had pain, discomfort, tightness, or pressure in your chest during or after exercise? 14. Do you get tired more quickly than your friends do during exercise?

15. Have you ever had racing of your heart or skipped heartbeats?

16. Have you had high blood pressure or high cholesterol?

17. Have you ever been told you have a heart murmur?

18. Has a physician ever denied or restricted your participation for sports or for any heart problems? 19. Have you ever had a head injury or concussion?

20. Have you ever been knocked out, become unconscious, or lost your memory? 21. Have you ever had an unexplained seizure?

22. Have you ever had numbness or tingling in your arms, hands, legs, or feet? 23. Have you ever become ill from exercising in the heat?

24. Do you cough, wheeze, or have trouble breathing during or after activity? 25. Do you have diabetes?

26. Do you have asthma or other lung disease?

27. Do you smoke?

28. Do you have or think you may have any bleeding disorders?

29. Have you had any other problems with injury, surgery, pain or swelling in muscles, tendons, bones, or joints? If yes to question 29, check appropriate blank and explain below.

220

__ Head __ Elbow __ Hip __ Foot __ Upper Arm __ Neck __ Forearm __ Thigh __ Ankle __ Shoulder __ Back __ Wrist __ Knee __ Shin/Calf __ Finger __ Chest __ Hand

Explain “Yes” answers here:

30. Do you have, or think you may have, any of the following conditions? Check

all that apply. Nausea, vomiting, or other gastrointestinal disorders Obstructive disease of the gastrointestinal tract, such as diverticulitis

or inflammatory bowel disease A history of disorders or impairment swallowing or of the gag reflex Any gastrointestinal surgery Explain “Yes” answers here:

31. Do you get frequent muscle cramps when exercising? Yes No

32. Do you or someone in your family have sickle cell trait or disease? Yes No

33. Have you ever experienced an exertional heat stroke or other Yes No significant heat illness? 34. Are you currently taking any prescription medications, pills, or an inhaler? Yes No For example: Albuterol, Prednisone, Naproxen, etc.

Explain “Yes” answers here:

35. Are you currently and regularly taking any non-prescription (over- the-counter) medications or pills? Yes No For example: Bayer, Prilosec, NyQuil, etc.

Explain “Yes” answers here:

221

36. Are you currently and regularly taking any vitamins, supplements, or other substances? Yes No For example: multi-vitamin, iron, diet pills, energy drinks, protein powder, etc.

Explain “Yes” answers here:

*FEMALES ONLY* 37. Is your menstrual cycle regular? Yes No

38. Are you on birth control? Yes No

39. Are you currently on your period? Yes No

40. What is the date of your last period?

I hereby state that, to the best of my knowledge, my answers to the above questions are complete and correct.

______Signature of Participant Printed Name of Participant Date

University of South Carolina Athletic Training Program Blatt PE Center Columbia, SC 29208

222

APPENDIX C. TAKE HOME INSTRUCTIONS

C.1. First Trial Email to Participants 4 Days Prior to Data Collection

Dear ______,

Thank you for participating in our study.

Starting ______we ask you to begin recording your diet and physical activity. First, you must initiate your account.

You will need to register on FoodProdigy. Use this online system to record all your daily intake of food and drinks as well as exercise and physical activity. This will allow us to look at your current habits throughout the study and mimic these for each trial. If you wish, you can receive a more detailed report of your information at the conclusion of the study.

To get started: 1. Go to www.foodprodigyonline.com/ui/registration and type in your email address. 2. Enter a unique Password. 3. Enter this Subscription ID: ______4. Go to www.foodprodigyonline.com. Use the email and password you registered with to log in to this account.

Use the FoodProdigy™ program to enter your diet and exercise information for 4 days. At the conclusion of the 4th day send your report to our co-investigator - Dr. Toni Torres- McGehee - using FoodProdigy. You will see a link that states e-mail Toni Torres- McGehee. We recommend you save a copy of the report for yourself so you can refer back to it.

Remember things to avoid during this time: Red meat Prescription or non-prescription NSAIDs Strenuous exercise

If you have any questions regarding FoodProdigy or completing the diet/activity log in general, please feel free to contact myself or Dr. Torres-McGehee.

Please to arrive to the lab on ______at ______to pick up your pre-trial information. Attached to this email are directions to the Public Health Research Center.

223

Sincerely, Dawn M. Emerson

Toni M. Torres-McGehee Associate Professor/Graduate AT Program Director University of South Carolina Blatt PE Center 218 Columbia, SC 29208

C.2. Reminder Email to Participants to Register and Complete Diet/Activity Log

Dear ______,

This is a reminder to register for FoodProdigy and begin today to record your daily diet and physical activity. You may stop recording ______and then send your report to Dr. Torres-McGehee.

Please let me know if you have any questions regarding FoodProdigy or your daily diet/activity log.

Sincerely, Dawn M. Emerson

C.3. Subsequent Trials Email to Participants 4 Days Prior to Data Collection

Dear ______,

Starting ______we ask you to begin recording your diet and physical activity. You must remember your login information for FoodProdigy (www.foodprodigyonline.com). Use the FoodProdigy program to enter all your daily intake of food and drinks as well as exercise and physical activity for 4 days. You will need to overwrite/clear your previous entries. At the conclusion of the 4th day send your report to our co-investigator - Dr. Toni Torres- McGehee - using FoodProdigy. You will see a link that states e-mail Toni Torres- McGehee. Remember things to avoid during this time: Red meat Prescription or non-prescription NSAIDs Strenuous exercise

224

If you have any questions regarding FoodProdigy or completing the diet/activity log in general, please feel free to contact myself or Dr. Torres-McGehee.

Please to arrive to the lab on ______at ______to pick up your pre-trial information. Attached to this email are directions to the Public Health Research Center. Contact us immediately if you must reschedule your appointment or if you have any questions or concerns.

Sincerely, Dawn M. Emerson

Toni M. Torres-McGehee Associate Professor/Graduate AT Program Director University of South Carolina Blatt PE Center 218 Columbia, SC 29208

C.4. Pre-Data Collection Take Home Instructions

Thank you for participating in our research investigation!

Arrive to the lab on ______, at your scheduled time ______. What should I wear? Athletic shorts, a t-shirt, socks, and a pair of athletic shoes Optional things I can bring? Change of clothes and a towel for after exercising A computer, book, or other material to use while you rest What should I eat or drink before I come? About 2 hours before coming to the lab you should eat a small meal (bagel, peanut butter and jelly, etc.) We also ask that you arrive hydrated by drinking water What things should I avoid during this time? Alcohol Non-prescription medications Energy supplements Dehydrating behaviors (sauna, diuretics, etc.) Exercise Changes in your sleep or eating habits What else do I need to do? Attached to this letter is:

225

1. An envelope with 2 pills Directions for taking pills:  Take 1st pill at ______Take each pill with a glass of water Do NOT take with food  Take 2nd pill at ______

2. A bag with a fecal test kit  Collect one fecal sample before you come to the lab Directions: Take card out of the bag and open the card. Apply a THIN smear of specimen on ONE window.

Do NOT cover the entire window.

Close the card and place back in bag. Throw away used stick.

3. A parking pass that you MUST bring with you to park at the Public Health Research Building. Use the handicap spaces on the left side of the parking lot behind the building.

Please contact us immediately if you need to reschedule your appointment or if you have any questions or concerns.

Sincerely, Dawn M. Emerson

C.5. Post-Data Collection Take Home Instructions

We have a few things for you to complete after you leave the lab today. 1. Continue recording your diet and activity using the online program until you collect your next fecal sample.

2. Using your fecal take home test kit, please collect your first fecal sample after leaving the lab Directions:

226

Take card out of bag and open the card. Apply a THIN smear of specimen to the SECOND window.

Do NOT cover the entire window.

Do NOT pull the tab or alter the card in any other way. Close the card and place back in bag

3. At the time of your fecal sample, complete the Gastrointestinal Symptom Index attached to this letter.

4. Return the fecal test kit and questionnaire by placing both items in the bag. Return the bag on ______at ______. At this time you will receive your payment.

Please contact us immediately if you need to reschedule your appointment or if you have any questions or concerns.

Thank you for your participation, Dawn M. Emerson

227

APPENDIX D. GASTROINTESTINAL SYMPTOM INDEXES D.1. GI Symptom Index during Exercise

Are you CURRENTLY experiencing GI symptoms?

0 No problem at all 1 2 3 4 5 6 7 8 9 Worst it has ever been

Reflux/Heartburn Left abdominal pain/stitch

Belching Right abdominal pain/stitch

Bloating Loose stool

Stomach pain/cramps Diarrhea

Vomiting Dizziness

Nausea Headache

Intestinal cramps Muscle cramps

Flatulence Urge to urinate

Urge to defecate

228

D.2. GI Symptom Index Pre- and Post-Exercise

Participant Number: ____ Trial Number: ____ Date: ______Time: ______

Directions: Please circle the number that corresponds to symptoms you are currently experiencing.

No Worst it problem has ever at all been Reflux/Heartburn 0 1 2 3 4 5 6 7 8 9 Belching 0 1 2 3 4 5 6 7 8 9 Bloating 0 1 2 3 4 5 6 7 8 9 Stomach pain/cramps 0 1 2 3 4 5 6 7 8 9 Vomiting 0 1 2 3 4 5 6 7 8 9 229 Nausea 0 1 2 3 4 5 6 7 8 9

Intestinal cramps 0 1 2 3 4 5 6 7 8 9 Flatulence 0 1 2 3 4 5 6 7 8 9 Urge to defecate 0 1 2 3 4 5 6 7 8 9 Left abdominal pain/stich 0 1 2 3 4 5 6 7 8 9 Right abdominal pain/stich 0 1 2 3 4 5 6 7 8 9 Loose stool 0 1 2 3 4 5 6 7 8 9 Diarrhea 0 1 2 3 4 5 6 7 8 9 Dizziness 0 1 2 3 4 5 6 7 8 9 Headache 0 1 2 3 4 5 6 7 8 9 Muscle cramps 0 1 2 3 4 5 6 7 8 9 Urge to urinate 0 1 2 3 4 5 6 7 8 9

APPENDIX E. DATA COLLECTION PROCEDURE CHECKLISTS

E.1. Information Session

30 minutes prior to participant arrival □ Set out: o Informed Consent Form o Second ICF signature sheet o Information Session sheet o Health and Injury History Questionnaire o Serving Size document o Pens o Computer . Ensure internet access . Logon to FoodProdigy o Stethoscope and BP cuff ̇ o If doing VO2max Test . Tanita scale . See V̇ O2max Procedures

Participant arrival □ Provide participant with ICF and second signature sheet □ Primary investigator reviews ICF and answers any questions □ Gather signed participant ICF □ Instruct participant to complete Health and Injury History Questionnaire □ Review information on diet/physical activity logging, show FoodProdigy online program o Review serving size document and give copy □ Assess participant’s resting HR and BP □ Gather participant’s self-reported height o Convert to cm (1 foot = 30.48 cm) ̇ □ If scheduled/able conduct VO2max test ̇ □ If not, schedule VO2max test □ Gather general information about available days/times to conduct data collection □ Provide Take Home Instructions if applicable

E.2. V̇ O2max Testing

(30 minutes prior to participant arrival) □ Ensure room is at appropriate thermoneutral temperature □ Obtain equipment:

230

o Bike o Heart rate strap o Heart rate monitor o RPE scale o Metronome o Pen o Tanita scale o “Information Session” sheet o Water bottle for after test □ Ensure Monark is working (batteries in computer), pedals, break system, etc.

Participant Arrival □ Ensure participant is dressed appropriately □ Verbally inquire/verify participant has not eaten immediately before coming, received an appropriate amount of sleep and has not performed strenuous exercise within 24 hours □ Request participant remove shoes and socks o Take weight measure □ Give participant HR strap and ensure working □ Allow participant to sit and rest while you complete the following: □ Briefly explain the purpose of the test in layman terms o Aerobic fitness level o Measurement of the maximal amount of oxygen one can consume o Relative measure of the amount of endurance work one can perform o Done in all professional endurance athletes ̇ o Describe range of values for VO2 max and what our criteria is o Encourage your subject to ask questions before you move on □ Explain the cycling protocol o Cycle to complete exhaustion (physical fatigue) o Minutes per stage o Incremental increases in resistance o Speed should be constant throughout the study (50rpm) o Describe the physiological measurement including heart rate and RPE . Rate of perceived exertion (RPE) • Describe this chart very clearly to participant . ‘During the exercise test we want you to pay close attention to how hard you feel the exercise work rate is. This feeling should reflect your total amount of exertion, and fatigue, combining all sensations and feelings of physical stress, effort and fatigue. Don’t concern yourself with factors such as leg pain, shortness of breath or exercise intensity, but try to concentrate on your total inner feeling of exertion. Try not to underestimate or overestimate your feelings of exertion. Be as accurate as you can.’ - ACSM o Repeat: Cycle to complete exhaustion (physical fatigue) □ Explain termination criteria

231

o Onset of angina or angina-like symptoms o Signs of poor perfusion: lightheadedness, ataxia, pallor, nausea, cold and clammy skin o Failure of HR to increase with increasing exercise intensity o Participant requests to stop o Failure of testing equipment o Participant cannot keep up cadence (50 RPM) during stage □ Encourage your subject to ask questions about this section before you move on □ Assess participant’s resting HR and blood pressure

Running the V̇ O2max test □ Position participant on bike o Seat height (5-10 degrees knee flexion at bottom position) o Handle bar setting o Make sure participant is comfortable before starting! o Record corresponding seat and bar height/position □ Explain clearly to subject that he/she will follow the metronome beeps for the cadence □ Ensure bike is set to 0 kp and allow participant to warm up for 2 minutes at set cadence □ Increase resistance (kp/watts) every 2 minutes as corresponding on the Information Sheet □ Record heart rate and RPE at the end of each stage □ You may provide encouragement to the participant, but this must be consistent between participants!

Ending V̇ O2 max Test □ Once participant reaches termination criteria, immediately record heart rate, RPE, and time □ Immediately decrease/remove resistance □ Allow participant to actively cool down for 3-5 minutes □ Monitor heart rate to ensure it decreases appropriately □ Provide water to participant

Criteria for reaching V̇ O2 max

□ Maximal heart rate within 10 beats of age-predicted max heart rate □ RPE > 17

Calculate V̇ O2max on data sheet Must be between ~ 40-50 ml/kg/min for males based on age and 31-40 ml/kg/min for females based on age and cycling vs running*

E.3. Pre-Trial Appointment

30 minutes before participant arrival □ Prepare Pre-Trial Take Home Instructions

232

o Fill out report time and date o Envelope with 2 pills o Fill out time to take each pill o Bag with fecal occult blood test and two sample collection sticks o Dated parking pass

Participant arrival □ Thoroughly review Take Home instructions with participant □ Answer any questions

E.4. Post-Trial Appointment

15 minutes before participant arrival □ Obtain participant payment □ Fill out receipt book

Participant arrival □ Obtain fecal occult blood test and GI Symptom Index □ Give participant money □ Have participant sign receipt book and provide participant with receipt □ Verify next trial appointment

E.5. Data Collection Session

60-90 minutes before participant arrival □ Verify chamber is set to appropriate temperature o Thermal: 35°C/95°F, 30% RH . If 32.5 then set humidity to 50% o Thermoneutral: 15°C/59°F, 30%RH . If in room 317 check temperature □ Set out following equipment: o Blood collection . Label 2 pink 6ml tubes for each blood time point (8 total) • Participant #, Trial #, pre • Participant #, Trial #, 80 min • Participant #, Trial #, post • Participant #, Trial #, 3hr post

#1 Trial 1 3hr Post

. Styrofoam cooler • Fill ¾ with ice o Gloves o Sugars

233

o Urine volume containers and 3 cups . Label cups: • Participant #, trial #, pre #6 • Participant #, trial #, post Trial 2 • Participant #, trial #, 3hr post o 3 gallon cooler . Fill with ice and water o 1 water bottle with cap, 1 water bottle without cap Graduated cylinder for water o rd o 3 pill o White plastic container with ice for urine o HR monitor and strap o Stop watch o RPE chart o GI symptom chart o GI Symptom Index o Refractometers . 2 clinical, 1 pen, and 1 digital o Pens and sharpies o Clipboards o Pre Post Data Collection sheet and Cycling Protocol sheet o 2.0ml microtubes #4 . Label 4 urine tubes Trial 3 • Participant #, Trial # . Label minimum 2 plasma tubes for each blood time point (minimum 8 total) • Participant #, Trial #, pre #1 • Participant #, Trial #, 80 min Trial 2 • Participant #, Trial #, post 3hr post • Participant #, Trial #, 3hr post o Distilled water o Disposable pipettes o Kestrel environmental monitor o Rectal temperature probe and reader o Tanita scale o Ice chest and tarp . Fill chest with ice and place with tarp in men’s or women’s locker room □ Bike o Calibrate Computrainer See Procedures OR o Calibrate Monark See Procedures o Set bike up to participant’s previously determined set-up □ Set up Kestrel in chamber (or room 317) to verify environment □ Calibrate refractometers o Clinical refractometers

234

. Using a disposable pipette, put 2 drops of distilled water onto prism . Close cover plate . Hold up to light . Adjust the correcting screw to make light/dark boundary coincide at 1.000 o Pen refractometer . Turn Pen on . Using a cup of distilled water, place end of pen into water • Ensure prism is covered by water . Wait for reading . If not 0, press ZERO to calibrate o Digital refractometer . Turn refractometer on . Using disposable pipette, pipette distilled water on prism . Wait for reading . If not 0, press ZERO to calibrate □ Check centrifuge and set up if necessary □ Prepare sugar solution o 5g lactulose, 5g sucrose, 2g mannitol powder mixed with 150ml water □ Prepare water to be consumed during exercise o 3.5ml/kg every 15 minutes o Measure from 3 gallon cooler into graduated cylinder o Pour into designated water bottle o Set aside bottle without cap to be used to refill participant bottle

Participant arrival □ Confirm participant o Consumed a light breakfast and water o Consumed 2 pills at assigned times o Dressed appropriately o Inquire if participant has been logging diet/activity and collected 1 fecal sample □ Primary investigator inserts venous catheter and takes 2 6ml vials for pre-trial measure Give participant HR strap and ensure monitor is reading HR □ rd □ Give participant 3 pill to consume with water □ Participant complete GI Symptom Index □ Assess pre-trial HR and BP □ Provide participant with urine cup and rectal probe o Instruct participant to provide pre-trial urine sample into cup, and to completely void bladder the rest of the way into the toilet o Instruct participant to insert rectal probe approximately 10cm past the anal sphincter (to marked line on probe) □ Measure urine specific gravity using clinical refractometer to ensure participant is < 1.020

235

□ Measure participants pre-trial body mass o Shirt and shorts o NO shoes and socks □ Measure pre-trial core body temperature □ Provide participant with sugar solution and instruct to consume within 5 minutes □ Explain fluid consumption during trial o “You will be provided with a designated amount of water in a bottle. We have superficially measured out this amount of water for you in order to ensure you maintain hydration. You are to consume the entire contents of the bottle, (their specific volume) ml, every 15 minutes. We encourage you to drink a little bit throughout the entire 15 minutes and to not wait till the end to consume the whole amount. At the end of each 15 minute increment we will provide you with more water.” o Answer any questions regarding this □ Take participant into chamber and orient him/her to the bike and room □ Explain cycle exercise o “Based on your VO2max test you will need to bike at an intensity that elicits a HR at their HR corresponding to 70% VO2max. We have set up the bike for you. This display shows your rpm and time. Here is the HR monitor. You will bike at this workload and HR for 80 minutes. Every 15 minutes we will ask you RPE (show scale) GI symptoms (explain scale), take BP and refill your water bottle. We will have the probe plugged into the monitor so we can continuously monitor your core temperature and we can also continuously monitor your HR. At the end of the 80 minutes we will collect 2 6ml vials of blood.” o Answer any questions up to this point. o “At the end of 80 minutes we will ask you to finish the last 10 minutes of the cycle exercise in an ‘all-out effort’. Consider it a sprint to the finish. You want to go as fast and far as you can in 10 minutes.” o Answer any questions up to this point. □ Record environment on Data Collection Cycle Protocol sheet o Check environment post-cycle, if different record on sheet

□ Co-investigators may run analysis and spin blood during cycle protocol o Follow Blood Mapping for appropriate procedures on handling, preparing, and freezing blood and plasma samples o Analyze urine samples with refractometers and record on Pre Post Data Collection sheet

Cycle Protocol □ Instruct participant to get on bike o Ensure participant is comfortable on the bike □ Allow participant to warm-up for 3 minutes o Answer any questions at this point and ensure their comfort o Check core temperature and HR are being monitored □ At 2.5 minutes measure time point 0

236

o Core temperature o HR o BP o RPE o GI symptoms □ Begin 70% VO2max/HR portion o 5 minutes record core temperature, HR, RPE, and GI symptoms . Remind participate to consume fluids o 10 minutes record core temperature, HR . Remind participate to consume fluids o 15 minutes record core temperature, HR, BP, RPE, and GI symptoms . Refill water bottle with designated fluid volume o 20 minutes record core temperature, HR . Remind participate to consume fluids o 25 minutes record core temperature, HR . Remind participate to consume fluids o 30 minutes record core temperature, HR, BP, RPE, and GI symptoms . Refill water bottle with designated fluid volume o 35 minutes record core temperature, HR . Remind participate to consume fluids o 40 minutes record core temperature, HR . Remind participate to consume fluids o 45 minutes record core temperature, HR, BP, RPE, and GI symptoms . Refill water bottle with designated fluid volume o 50 minutes record core temperature, HR . Remind participate to consume fluids o 55 minutes record core temperature, HR . Remind participate to consume fluids o 60 minutes record core temperature, HR, BP, RPE, and GI symptoms . Refill water bottle with designated fluid volume o 65 minutes record core temperature, HR . Remind participate to consume fluids o 70 minutes record core temperature, HR . Remind participate to consume fluids o 75 minutes record core temperature, HR, BP, RPE, and GI symptoms . Refill water bottle with designated fluid volume o 80 minutes record core temperature, HR, RPE, and GI symptoms . Take blood sample • Flush catheter with saline . Record starting distance o 85 minutes record core temperature, HR o 90 minutes record core temperature, HR, BP, RPE, and GI symptoms . Record end distance □ Allow participant to actively cool down for 5 minutes o Take post-cycle blood measure . Flush catheter

237

o Allow to consume fluid and refill bottle if necessary o Monitor and record HR and core temperature □ At end of 5 minutes remove from bike □ Instruct participant to towel off any sweat and remove shoes and socks □ Weigh participant □ Allow to rest for 10 minutes o Record HR and core temperature o Instruct to complete post- GI Symptom Index □ Provide urine cup and urine volume container o Instruct participant to provide small sample in urine cup o Instruct participant to completely void urine into the large urine container . “Please Do NOT void into the toilet/urinal” o Place urine volume container in white tub with ice □ Participant may remove rectal thermometer o If participant chooses to remove it, remind them a final core temperature must be taken at the end of the 3 hours □ Participant may change clothes at this point o Instruct them to take care to not pull out the catheter o Instruct them to complete this as efficiently as possible to avoid an extended period in the locker room/restroom □ While participant is gone o Prepare fluid to be consumed during rest (provide amount enough ½ hour) □ Once participant returns □ Once finished participant rests for 3 hours o Remind participant to consume fluids regularly to promote hydration and urine volume o Instruct participant that any time he/she needs to void urine it must be done in the urine volume container o He/she is permitted to work, read, or watch something to occupy themselves over the next 3 hours; however, he/she cannot leave and should not be up being active □ Every 1 hour venous catheter should be flushed and water bottle refilled □ Approximately ten minutes before the end of the rest o Provide GI Symptom Index o Review Post-Trial Take Home Instructions . Provide GI Symptom Index . Set up return appointment o Obtain 2 6ml vials of blood . Remove catheter o Weigh o Core temperature (unless not inserted, then must reinsert and measure at end of rest) □ At end of rest o Obtain urine sample in urine cup and void any remaining urine into urine volume container o Instruct participant to remove rectal probe

238

□ Answer any questions and finalize session

Post-trial □ Centrifuge all blood o Follow Blood Mapping □ Record total urine volume o Pipette aliquots of urine into the 4 microtubes to be frozen □ Record total fluid volume consumed □ Ensure all blood and urine samples are in freezer and appropriately labeled □ Ensure all biohazard/sharps are appropriately disposed of □ Clean all equipment o Water bottles o Wipe down bike o Rectal probe o Counters and tables o Cooler and cylinder (end of day) □ Put away all equipment o Kestrel o Scale □ Ensure data sheets are all in box □ Turn off all equipment and lock up all labs

Miscellaneous □ Avoid providing food unless participant absolutely is in need of food. In these instances, attempt to provide non-sucrose containing foods

In Case of Emergency

□ Immediately notify primary investigator if not currently involved with incident □ Cardiac o Call 911, obtain AED from room 317 o Begin CPR □ Exertional heat stroke o Confirm heat stroke . CNS dysfunction and core temperature above 104°F/40°C o Immediately remove from environmental chamber and into locker room o Lay out tarp and place participant on tarp o Turn on cold water in shower o Dump ice over participant o Monitor core temperature and HR □ As soon as possible, call supervising physician o Dr. Brian Kiesler □ Notify supervising faculty advisor o Dr. Mark Davis □ Complete incident report.

239

APPENDIX F. ADDITIONAL TABLES AND FIGURES

38.8 Control 38.6 Npx

38.4 Heat NpxHeat 38.2 ° C) 38.0

37.8

240 37.6

37.4 Core Temperature ( Core Temperature

37.2

37.0

36.8

36.6 Pre 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 5 10 15 30 45 60 75 90 105 Cycling (min) Rest (min)

Figure F.1. Male Mean Core Temperature during Cycling and Rest for Experimental Conditions No significant differences between conditions at any time point

38.8 Control 38.6 Npx

38.4 Heat NpxHeat 38.2 ° C) 38.0

37.8

37.6

Core Temperature ( Core Temperature 37.4 241

37.2

37.0

36.8

36.6 Pre 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 5 10 15 30 45 60 75 90 105

Cycling (min) Rest (min)

Figure F.2. Female Mean Core Temperature during Cycling and Rest for Experimental Conditions No significant differences between conditions at any time point.

38.6 Male Female 38.4 a

38.2

38.0 ° C)

37.8

a 37.6 a a 37.4 a a a a

242 a Core Temperature ( Core Temperature

37.2

37.0

36.8

36.6 Pre 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 5 10 15 30 45 60 75 90 105

Cycling (min) Rest (min) Figure F.3. Mean Core Temperature during Cycling and Rest for Gender. aSignificant difference between gender (P < 0.05).

5.2 Control

4.8 a Npx Heat 4.4 b NpxHeat 4.0 3.6 3.2 2.8 6 (pg/ml) 6 - 2.4 IL 243 2.0

1.6 1.2 0.8 0.4 0.0 Pre Post 3 hr Post

Figure F.4. Mean IL-6 Pre-, Post-, and 3 Hours Post-Cycling for Males. N = 4. Significant main effect for each condition (P < 0.04). aSignificantly greater than pre (P = 0.014) and 3 hr post (P = 0.038). bSignificantly greater than pre (P = 0.042).

5.2 Control 4.8 Npx Heat 4.4 NpxHeat 4.0 3.6 3.2 2.8 6 (pg/ml) 6 - 2.4 IL 244 2.0 a 1.6 b 1.2 0.8 0.4 0.0 Pre Post 3 hr Post

Figure F.5. Mean IL-6 Pre-, Post-, and 3 Hrs Post-Cycling for Females. N = 3. Significant main effect for Control (P = 0.022) and Npx (P = 0.017). aSignificantly greater than 3 hr (P = 0.046). bSignificantly greater than pre (P = 0.015).

Table F.1. Fluid Volume and Urine Volume for Gender by Experimental Conditions (M + SD)

Fvol (L) Fvol (ml/kg) Uvol (L) Exercise Total Exercise Total Aggregate Males 1.7 + 0.7a 2.5 + 1.3a 19.0 + 6.5 27.5 + 11.9 1.1 + 0.6 Females 1.1 + 0.5 1.7 + 0.7 16.3 + 5.9 24.1 + 7.2 0.9 + 0.4 Control Males 1.3 + 0.5 1.9 + 0.7 14.1 + 3.4 20.3 + 5.7 1.2 + 0.8 Females 1.0 + 0.3 1.5 + 0.6 14.4 + 5.5 22.8 + 8.0 1.1 + 0.5 Npx 245 Males 1.9 + 1.0b 2.8 + 1.8 20.7 + 8.8 30.8 + 16.8 1.3 + 0.6 Females 0.9 + 0.5 1.4 + 0.7 12.1 + 5.0 20.1 + 7.1 0.8 + 0.5 Heat Males 1.7 + 0.7 2.5 + 1.2 18.8 + 5.3 27.4 + 10.1 1.1 + 0.6 Females 1.4 + 0.4 1.9 + 0.7 20.7 + 4.6 27.2 + 6.1 0.8 + 0.3 HeatNpx Males 2.0 + 0.6a 3.0 + 1.4 22.5 + 5.4 32.0 + 12.8 0.9 + 0.1 Females 1.3 + 0.5 1.9 + 0.9 17.9 + 5.9 26.4 + 7.4 0.7 + 0.3

aSignificantly higher than females (P < 0.05). bApproached significance (P = 0.055).

a) Males b) Females 180 180 Control a

170 170 Npx b 160 160 Heat NpxHeat 150 150 140 140 c 130 130 120 120 110 110 Systolic Blood Pressure (mmHg) Pressure Blood Systolic

246 100 100

Pre Post 3 hr Post Pre Post 3 hr Post Figure F.6 Mean Systolic BP Pre-, Post-, and 3 Hrs Post-Exercise for Experimental Conditions by Gender. a Significant main effect over time (F2,40 = 37.0, P < 0.001) for males (a) and females (b). Significantly higher than b female Npx post (F1,10 = 8.5, P = 0.017). Significantly higher than female NpxHeat post (F1,10 = 5.8, P = 0.043). c Significantly higher than female Heat 3 hrs (F1,10 = 6.9, P = 0.028).

Table F.2. Diet and Exercise Overall and for Gender (M + SD).

Aggregate (N = 22) Males (N = 14) Females (N = 8)

Calories (kcal) 1522.2 + 388.0 1660.1 + 379.6b 1280.9 + 281.8

Protein (g) 67.3 + 21.6 75.9 + 19.6b 52.3 + 16.6

Fat (g) 48.1 + 19.9 51.4 + 22.9 42.3 + 12.5

Carb (g) 194.5 + 62.7 206.7 + 73.0 173.0 + 32.9

Sodium (mg) 2401.1 + 759.2 2650.9 + 853.7b 1964.0 + 180.0

Exercisea 260.4 + 78.9 226.0 + 29.1 303.5 + 105.0 247 N = number of logs reported (22/44). aBased on calories burned during activity 24 hrs prior to data collection. All reported activity categorized as low-sedentary. bSignificantly greater than females (P < 0.04).

Table F.3. Rate of Perceived Exertion during Cycling for Gender by Experimental Condition (M + SD) Cycle time Aggregate Control Npx Heat NpxHeat (min) Male Female Male Female Male Female Male Female Male Female

0 8 + 2a 10 + 2 8 + 2 9 + 2 8 + 3 10 + 3 8 + 3 10 + 3 7 + 1b 9 + 2

15 9 + 3a 11 + 2 10 + 3 12 + 1 10 + 2 11 + 3 8 + 3 11 + 2 9 + 3 11 + 2

30 10 + 3 12 + 2 11 + 4 11 + 2 11 + 2 11 + 3 9 + 3 12 + 1 10 + 3 12 + 2

45 11 + 3 12 + 2 11 + 3 13 + 1 12 + 2 11 + 3 10 + 2 12 + 1 11 + 3 12 + 2

60 11 + 2 12 + 2 11 + 3 12 + 1 12 + 2 11 + 3 11 + 2 12 + 2 12 + 2 12 + 1

75 12 + 2 12 + 2 12 + 3 13 + 2 12 + 3 12 + 3 11 + 2 13 + 1 11 + 2 12 + 2

248 80 12 + 2 13 + 2 12 + 2 14 + 2 12 + 3 13 + 2 12 + 2 13 + 1 12 + 2 13 + 2

90 19 + 2a 18 + 2 19 + 2 18 + 2 20 + 1 18 + 2 19 + 2 18 + 2 19 + 2 18 + 2

No significant difference between experimental conditions within gender. aSignificantly different than females (P < 0.03). bSignificantly less than females (P = 0.03).

Table F.4. Distance and Max Heart Rate during Cycling for Gender by Experimental Condition (M + SD)

Distance (miles) Max HR (bpm) Aggregate Males 3.2 + 0.7 177.2 + 17.1 Females 2.8 + 0.9 177.2 + 15.0 Control Males 3.4 + 0.8 175.0 + 16.6 Females 2.9 + 0.9 176.6 + 12.7 Npx Males 3.5 + 0.7 179.5 + 15.1 Females 3.0 + 1.0 172.2 + 15.5 Heat Males 3.0 + 0.7 176.3 + 20.2 Females 2.7 + 1.0 179.6 + 17.6 NpxHeat Males 3.0 + 0.8 178.0 + 20.3 Females 2.5 + 0.9 180.2 + 17.2

249

Table F.5. Mean, Maximum, and Serious (> 4) Gastrointestinal Symptom Scores for Experimental Conditions Upper Lower Systemic Condition Max Mean + SD Serious Max Mean + SD Serious Max Mean + SD Serious Control Pre 2 0.1 + 0.1b - 2 0.0 + 0.1 - 1 0.1 + 0.1f - Post 2 0.2 + 0.2 - 2 0.1 + 0.1 - 8 0.9 + 0.9 6% 3hr Post 5 0.2 + 0.3 3% 3 0.2 + 0.2d,e - 5 0.7 + 0.7j 6% Day Postk - - - 5 0.3 + 1.0 3% 2 0.2 + 0.5 - Npx Pre 2 0.1 + 0.1 - 1 0.0 + 0.1 - 2 0.2 + 0.3 - Post 2 0.1 + 0.2 - 2 0.0 + 0.1 - 7 0.6 + 0.6g 5% 3hr Post 2 0.0 + 0.1 - 1 0.0 + 0.1 - 2 0.1 + 0.2 - Day Postk - - - 4 0.2 + 0.8 - - - -

250 Heat a Pre 0 0.0 + 0.0 - 1 0.0 + 0.0 - 3 0.2 + 0.3 - Post 2 0.3 + 0.3c - 2 0.1 + 0.1 - 8 0.7 + 0.9h 3% 3hr Post 1 0.0 + 0.1a - 2 0.1 + 0.1 - 6 0.4 + 0.6 3% Day Postk 6 1.3 + 2.5 18% 6 1.9 + 2.4 6% 6 1.3 + 2.2 5% NpxHeat Pre 2 0.1 + 0.2 - 1 0.0 + 0.0 - 5 0.3 + 0.6 3% Post 2 0.2 + 0.3 - 1 0.0 + 0.1 - 3 0.3 + 0.4i - 3hr Post 4 0.1 + 0.2 - 2 0.1 + 0.1 - 3 0.2 + 0.2 - Day Postk 3 0.1 + 0.6 - 7 0.7 + 2.1 12% 2 0.1 + 0.5 - aSignificantly less than Heat post (Z = -2.3, P < 0.019). bSignificantly greater than Heat pre (Z = -2.1, P = 0.038). cSignificantly greater than Npx post (Z = -2.2, P = 0.027). dSignificantly greater than Npx 3hr (Z = -2.0, P = 0.048), HeatNpx 3hr (Z = -2.4, P = 0.014). eSignificantly greater than Control pre (Z = -2.4, P = 0.016), post (Z = -2.1, P = 0.039). fSignificantly less than post (Z = - 2.4, P = 0.018), 3hr post (Z = -2.2, P = 0.026). gSignificantly greater than 3hr (Z = -2.1, P = 0.035). hSignificantly greater than pre (Z = -2.0, P = 0.042). iSignificantly less than Control post (Z = -2.0, P = 0.046). jSignificantly greater than Npx 3hr (Z = -2.4, P = 0.018), Heat 3hr (Z = -2.0, P = 0.042). kVarying sample size (n = 3-5) and not included in Wilcoxon analysis.

Table F.6. Mean and Serious (> 4) Gastrointestinal Symptom Scores for Experimental Conditions during Exercise

Upper Lower Systemic Condition Mean + SD Serious Mean + SD Serious Mean + SD Serious Control 0.3 + 0.6 1% 0.4 + 0.8 - 0.3 + 0.4 10% Npx 0.4 + 0.7 1% 0.1 + 0.3 - 0.2 + 0.3 10% Heat 0.6 + 1.0 3% 0.3 + 0.6 - 0.1 + 0.3 7% NpxHeat 0.3 + 0.9 2% 0.3 + 0.7 - 0.0 + 0.8a 1%

aSignificantly less than Control (Z = -2.0, P = 0.046). 251